Sterile chromatography and manufacturing processes

ABSTRACT

Provided herein are methods of performing chromatography with gamma-irradiated chromatography resin that include providing a chromatography column including a gamma-irradiated chromatography resin; performing a first cycle of chromatography through the column, where the cycle includes exposing the chromatography resin to a denaturing buffer; and performing at least one additional cycle of chromatography through the column. Also provided are integrated, closed or substantially closed, and continuous processes for manufacturing of a recombinant protein that include the use of at least one chromatography column including gamma-irradiated chromatography resin, where the gamma-irradiated chromatography resin is exposed to denaturing buffer during each cycle in the process, and reduced bioburden buffer is used in the process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/598,450, filed on Jan. 16, 2015, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/928,906,filed on Jan. 17, 2014, which is incorporated by reference in itsentirety.

TECHNICAL FIELD

This invention relates to methods of biotechnology and thebiomanufacturing of recombinant proteins.

BACKGROUND

Mammalian cells including a nucleic acid that encodes a recombinantprotein are often used to produce therapeutically or commerciallyimportant proteins. In the current environment of diverse productpipelines, biotechnology companies are increasingly driven to developinnovative solutions for highly flexible and cost-effectivemanufacturing of therapeutic protein drug substances. One of thestrategies for efficiently isolating recombinant proteins is throughprocesses that include continuous chromatography (e.g., using a closedsystem). One known limitation of continuous chromatography is thepresence of contaminating agents in the system (e.g., increasedbioburden), which results in a contaminated product, a reduction in theproduction yield, and a decrease in the flow-rate (or increase in thepressure) in the system. For example, the increased bioburden within asystem can result in the complete shut down of the system.

SUMMARY

The present invention is based, at least in part, on the discovery thatgamma-irradiation of chromatography resin results in a decrease in thebinding capacity of the resin (e.g., a steady decrease in the bindingcapacity of the resin over multiple cycles of chromatography), and thatthe binding capacity of the gamma-irradiated resin can be recovered byexposing the resin to a denaturing buffer. In view of this discovery,provided herein are methods of performing chromatography withgamma-irradiated chromatography resin that include providing achromatography column including a gamma-irradiated chromatography resin;performing a first cycle of chromatography through the column, where thecycle includes exposing the chromatography resin to a denaturing buffer;and performing at least one additional cycle of chromatography throughthe column. Also provided are integrated, closed or substantiallyclosed, and continuous processes for manufacturing of a recombinantprotein that include the use of at least one chromatography columnincluding gamma-irradiated chromatography resin, where thegamma-irradiated chromatography resin is exposed to denaturing bufferduring each cycle in the process, and reduced bioburden buffer is usedin the process. Any of the methods and processes described herein can bereduced bioburden, sterile, aseptic, or absolutely sterile methods orprocesses (as defined herein). Any of the methods and processesdescribed herein can be a combination of aseptic and reduced bioburden,sterile, or absolutely sterile.

Provided herein are methods of performing chromatography withgamma-irradiated chromatography resin that include: (a) providing achromatography column containing a gamma-irradiated chromatographyresin; (b) performing a first cycle of chromatography through thecolumn, wherein the cycle includes exposing the chromatography resin toa denaturing buffer; and (c) performing at least one additional cycle ofchromatography through the column. In some embodiments of these methods,performing the cycles in (b) and/or (c) includes the steps of: (a)capturing a recombinant protein by exposing the chromatography resinwith a liquid containing a recombinant protein; (b) washing thechromatography resin by exposing the chromatography resin with a washbuffer; (c) eluting the recombinant protein by exposing thechromatography resin with an elution buffer; and (d) regenerating thechromatography resin by exposing the chromatography resin to thedenaturing buffer.

In some examples of any of the methods described herein, the liquidcontaining a recombinant protein is a liquid culture medium. In someembodiments of any of the methods described herein, the cycles in (b)and (c) are performed using a closed and integrated system. In someexamples of any of the methods described herein, the buffer is reducedbioburden buffer (e.g., a reduced bioburden buffer prepared byfiltration).

In some examples of any of the methods described herein, the denaturingbuffer includes one or more of urea, guanidine hydrochloride, andTriton™ X-100. In some embodiments of any of the methods describedherein, the cycle of (b) further includes exposing the chromatographyresin to wash buffer including about 0.5 M to about 1.5 M sodiumhydroxide following exposure to the denaturing buffer. In someembodiments where an affinity chromatography resin including a proteinligand is used, the cycle of (b) further includes exposing thechromatography resin to wash buffer including about 1 mM to about 100 mMsodium hydroxide. In some examples of any of the methods describedherein, the column is part of a multi-column chromatography system(MCCS) (e.g., a periodic counter current chromatography system (PCCS).

In some embodiments of any of the methods described herein, thechromatography resin is anionic exchange chromatography resin, cationicexchange chromatography resin, size exclusion chromatography resin,hydrophobic interaction chromatography resin, affinity chromatographyresin, or any combination thereof. In some examples, the chromatographyresin is anionic exchange chromatography resin.

In some examples of any of the methods described herein, thechromatography resin has been treated with a dose of gamma-irradiationbetween about 10 kGy to about 40 kGy (e.g., between about 15 kGy toabout 35 kGy, or between about 20 kGy to about 30 kGy). In someembodiments of any of the methods described herein, four or more (e.g.,nine or more, fourteen or more, nineteen or more, twenty-four or more,twenty-nine or more, or thirty-nine or more) additional cycles ofchromatography are performed. In some examples of any of the methodsdescribed herein, step (c) is performed over a period of at least 4 days(e.g., at least 5 days, at least 7 days, at least 14 days, or at least28 days). In some examples of any of the methods described herein, therecombinant protein is a recombinant therapeutic protein.

Also provided are integrated, closed or substantially closed, andcontinuous processes for manufacturing of a purified recombinant proteinthat include: (a) providing a liquid culture medium including arecombinant protein that is substantially free of cells; and (b)continuously feeding the liquid culture medium into a multi-columnchromatography system (MCCS) including at least one chromatographycolumn containing gamma-irradiated chromatography resin, wherein thechromatography resin is exposed during each cycle to denaturing buffer;where the process utilizes reduced bioburden buffer, is integrated, andruns continuously from the liquid culture medium to an eluate from theMCCS that is the purified recombinant protein. In some embodiments ofthese processes, the MCCS performs at least two different unitoperations. In some examples of any of the processes described hereinthe MCCS involves column switching. In some embodiments of any of theprocesses described herein, all of the columns in the MCCS containgamma-irradiated chromatography resin.

In some examples of any the processes described herein, the MCCSperforms the unit operations of capturing the recombinant protein andinactivating viruses, or the unit operations of capturing and purifyingthe recombinant protein. In some embodiments of any of the processesdescribed herein, the MCCS is a periodic counter current chromatographysystem. In some examples of any of the processes described herein, theMCCS includes a plurality of columns for affinity chromatography, cationexchange chromatography, anion exchange chromatography, size exclusionchromatography, or hydrophobic interaction chromatography, or anycombination thereof. In some embodiments of any of the processesdescribed herein, where the MCCS includes a column for affinitychromatography, and the affinity chromatography is performed with acapture mechanism selected from the group consisting of: proteinA-binding capture mechanism, substrate-binding capture mechanism,antibody- or antibody fragment-binding capture mechanism,aptamer-binding capture mechanism, and cofactor-binding capturemechanism. In some examples of any of the processes described herein,the affinity chromatography is performed with a protein-A bindingcapture mechanism, and the recombinant protein is an antibody or anantibody fragment. In some examples of any of the processes describedherein, the recombinant protein is a therapeutic recombinant protein.Some embodiments of any of the processes described herein furtherinclude formulating the purified recombinant protein into apharmaceutical composition. In some embodiments of any of the processesdescribed herein, the denaturing buffer includes one or more of urea,guanidine hydrochloride, and Triton™ X-100. In some examples of any ofthe processes described herein, the chromatography resin is exposed towash buffer including about 0.5 M to about 1.5 M sodium hydroxidefollowing exposure to denaturing buffer in each cycle.

Also provided are integrated, closed or substantially closed, andcontinuous processes for manufacturing of a recombinant protein thatinclude: (a) providing a liquid culture medium including a recombinantprotein that is substantially free of cells; (b) continuously feedingthe liquid culture medium into a first multi-column chromatographysystem (MCCS1); (c) capturing the recombinant protein from the liquidculture medium using the MCCS1; (d) producing an eluate from the MCCS1that includes the recombinant protein and continuously feeding theeluate into a second multi-column chromatography system (MCCS2); (e)continuously feeding the recombinant protein from the eluate into theMCCS2 and subsequently eluting the recombinant protein to therebyproduce the purified recombinant protein, where: the process utilizesreduced bioburden buffer, is integrated, and runs continuously from theliquid culture medium to the purified recombinant protein, and at leastone column in the MCCS1 and/or MCCS2 is a chromatography columncontaining gamma-irradiated chromatography resin and the chromatographyresin is exposed to denaturing buffer during each cycle in the process.In some examples of any of the processes described herein, the MCCS1and/or the MCCS2 performs at least two different unit operations. Insome examples of any of the processes described herein, the use of theMCCS1 or the MCCS2, or both, involves column switching.

In some embodiments of any of the processes described herein, the MCCS1further performs the unit operations of capturing the recombinantprotein and inactivating viruses. In some embodiments of any of theprocesses described herein, the MCCS2 performs the unit operations ofpurifying and polishing the recombinant protein. In some examples of anyof the processes described herein, the MCCS1 and/or MCCS2 utilizes atleast two chromatography columns. In some examples of any of theprocesses described herein, all of the chromatography column(s) in MCCS1and MCCS2 are chromatography columns containing gamma-irradiatedchromatography resin. In some examples of any of the processes describedherein, the MCCS1 is a first periodic counter current chromatographysystem (PCCS1). In some embodiments of any of the processes describedherein, the capturing is performed using affinity chromatography, cationexchange chromatography, anion exchange chromatography, or sizeexclusion chromatography, hydrophobic interaction chromatography, or anycombination thereof. In some examples of any of the processes describedherein, the capturing is performed using affinity chromatography with acapture mechanism selected from the group of: protein A-binding capturemechanism, substrate-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, aptamer-binding capture mechanism,and cofactor-binding capture mechanism. In some embodiments of any ofthe processes described herein, the affinity chromatography is performedwith a protein A-binding capture mechanism, and the recombinant proteinis an antibody or an antibody fragment.

In some embodiments of any of the processes described herein, the MCCS2is a second periodic counter current (PCCS2) chromatography system. Insome examples of any of the processes described herein, the recombinantprotein is a therapeutic recombinant protein. Some embodiments of any ofthe processes described herein further include formulating the purifiedrecombinant protein into a pharmaceutical composition. In some examplesof any of the processes described herein, the process is performedcontinuously for a period of at least 4 days (e.g., at least 5 days, atleast 7 days, at least 14 days, or at least 28 days).

In some embodiments of any of the processes described herein, thechromatography resin is anionic exchange chromatography resin, cationicexchange chromatography resin, size exclusion chromatography resin,hydrophobic interaction chromatography resin, affinity chromatographyresin, or any combination thereof. In some examples of any of theprocesses described herein, the chromatography resin is anionic exchangechromatography resin. In some examples of any of the processes describedherein, the chromatography resin has been treated with a dose ofgamma-irradiation between about 10 kGy to about 40 kGy (e.g., betweenabout 15 kGy to about 35 kGy, or between about 20 kGy to about 30 kGy).In some embodiments of any of the processes described herein, thedenaturing buffer includes one or more of urea, guanidine hydrochloride,and Triton™ X-100. In some embodiments of any of the processes describedherein, the chromatography resin is exposed to a wash buffer includingabout 0.5 M to about 1.5 M sodium hydroxide following exposure to thedenaturing buffer. In some embodiments of any of the processes describedherein where the chromatography resin is an affinity resin with aprotein ligand (e.g., a protein A ligand), the chromatography resin isexposed to a wash buffer including between about 1 mM to about 100 mMsodium hydroxide following exposure to the denaturing buffer.

As used herein, the word “a” before a noun represents one or more of theparticular noun. For example, the phrase “a chromatography column”represents “one or more chromatography columns.”

The term “gamma-irradiated chromatography resin” means a chromatographyresin that has been exposed to gamma-irradiation. For example, agamma-irradiated chromatography resin can be a chromatography resinexposed to an amount of gamma-irradiation sufficient to reduce thebioburden of the chromatography resin. In some examples, agamma-irradiated chromatography resin has been exposed to a dose ofbetween about 1 kGy to about 15 kGy, a dose of between about 1 kGy toabout 20 kGy gamma-irradiation, a dose of between about 1 kGy to about25 kGy gamma-irradiation, a dose of between about 1 kGy to about 30 kGygamma-irradiation, or a dose of between about 1 kGy to about 35 kGygamma-irradiation. A gamma-irradiated chromatography resin can have asterility assurance level of about or less than 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸,1×10⁻⁹, or 1×10⁻¹⁰. Exemplary methods for gamma-irradiating achromatography resin are described herein. Additional methods forgamma-irradiating a chromatography resin are known in the art.

The term “chromatography column containing, including, or comprising agamma-irradiated chromatography resin” means a chromatography columnincluding a gamma-irradiated chromatography resin (as defined herein).For example, such a chromatography column can include a gamma-irradiatedchromatography resin that has been exposed to a dose of between about 1kGy to about 15 kGy, a dose of between about 1 kGy to about 20 kGygamma-irradiation, a dose of between about 1 kGy to about 25 kGygamma-irradiation, a dose of between about 1 kGy to about 30 kGygamma-irradiation, or a dose of between about 1 kGy to about 35 kGygamma-irradiation. For example, such a chromatography column can containa gamma-irradiated chromatography resin having a sterility assurancelevel of about or less than 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹, or 1×10⁻¹⁰.In some embodiments, the chromatography column containing, including, orcomprising a gamma-irradiated chromatography resin has a reducedbioburden, or is sterile, absolutely sterile, or aseptic (as definedherein) (i.e., the interior surfaces and contents of the chromatographycolumn containing, including, or comprising a gamma-irradiatedchromatography resin has reduced bioburden, is sterile, is absolutelysterile, or is aseptic). In some embodiments, the chromatography columncontaining, including, or comprising a gamma-irradiated chromatographyresin is aseptic and has a reduced bioburden, is sterile, or isabsolutely sterile.

The term “denaturing buffer” means a liquid including a sufficientamount of one or more chemical agents (e.g., detergent(s), reductant(s),acid(s), base(s), chaotropic agent(s), organic solvent(s), orcrosslinking agent(s), or any combination thereof) that result in thedenaturation of a protein. Non-limiting examples of denaturing buffersare described herein. Additional examples of denaturing wash buffers areknown in the art. Methods for detecting protein denaturation are alsowell-known in the art (e.g., detecting protein denaturation directly(e.g., spectroscopy) or indirectly (e.g., through protein activity(e.g., enzymatic activity or protein binding activity) assay(s)).Non-limiting examples of methods for detecting protein denaturation aredescribed herein. In any of the methods or processes described herein, adenaturing buffer can be used to regenerate a chromatography columnincluding a gamma-sterilized chromatography resin (e.g., any of thechromatography resin or combination or chromatography resins describedherein).

The term “reduced bioburden buffer” means a treated (e.g., filtered,autoclaved, or gamma-irradiated) liquid (e.g., a treated bufferedsolution) that has a level of self-replicating biological contaminatingagent(s) that is less than the level of self-replicating biologicalcontaminating agent(s) found in an identical untreated liquid.Non-limiting examples of self-replicating biological contaminants can bebacteria (e.g., Gram-positive or Gram-negative bacteria, or bacterial orfungal spores), mycobacteria, viruses (e.g., a vesivirus, a Cache Valleyvirus, a parvovirus, a herpes virus, and a bunyavirus), parasites,fungi, yeast, and protozoa. For example, a reduced bioburden buffer canhave a sterility assurance level of about or less than 1×10⁻⁶, 1×10⁻⁷,1×10⁻⁸, 1×10⁻⁹, or 1×10⁻¹⁰.

“Absolute sterility” or “absolutely sterile” are terms used to describea composition or process that is/are completely free of self-replicatingbiological contaminants. For example, the term can apply to agamma-irradiated chromatography resin, the interior surface and contents(e.g., chromatography resin) of a chromatography column, and/or abuffer. An absolutely sterile composition or process can be clean (asthat term is known in the art).

“Sterile” or “sterility” are terms used to describe a composition orprocess that have a sterility assurance level of about or less than1.0×10⁻⁶ (e.g., about or less than 1.0×10⁻⁷, about or less than1.0×10⁻⁸, about or less than 1.0×10⁻⁹, or 1×10⁻¹⁰). The determination ofwhether a composition or process is sterile can be tested using a numberof validated production processes known in the art. For example, asterile composition or process can be completely free of viableself-replicating biological contaminants (e.g., any of theself-replicating biological contaminants described herein). A sterilecomposition or process can also be clean (as that term is known in theart).

The term “sterilization” means a validated process used to render acomposition sterile (as defined herein). The inactivation rate ofresistant indicator self-replicating biological contaminants (e.g.,bacteria) during a treatment process can be measured in order todetermine whether sterility (as defined herein) has been achieved for acomposition.

The term “sterility assurance level” or “SAL” is art-known and means alevel of confidence of achieving absolute sterility within a batch oftreated units. The probability is usually calculated based on theresults of inactivation studies performed during validation andexpressed in the form of 1×10^(−n).

The term “aseptic” is used to describe a composition or process that isfree of disease-causing or symptom-causing self-replicating biologicalcontaminants and/or proteins (e.g., any of the self-replicatingbiological contaminants described herein, toxins (e.g., endotoxins), orinflammatory proteins). An aseptic composition or process can also beclean (as that term is known in the art).

The term “cycle of chromatography” or “chromatography cycle” is a termof art and means all the steps performed in a single round ofchromatography using a single chromatography column. For example, acycle of chromatography can include a step of equilibrating achromatography column with a buffer, passing a sample including arecombinant protein through the chromatography column, eluting therecombinant protein from the chromatography column, and washing thechromatography column by passing a denaturing buffer through the column.Additional examples of steps performed in a cycle of chromatography aredescribed herein. Further examples of steps performed in a cycle ofchromatography are also well known in the art.

The term “unit operation” is a term of art and means a functional stepthat can be performed in a process of purifying a recombinant proteinfrom a liquid culture medium. For example, a unit of operation can befiltering (e.g., removal of contaminant bacteria, yeast, viruses, ormycobacteria, and/or particulate matter from a fluid including arecombinant protein), capturing, epitope tag removal, purifying, holdingor storing, polishing, virus inactivating, adjusting the ionicconcentration and/or pH of a fluid including the recombinant protein,and removing unwanted salts.

The term “capturing” means a step performed to partially purify orisolate (e.g., at least or about 5%, e.g., at least or about 10%, 15%,20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orat least or about 95% pure by weight) and concentrate a recombinantprotein (e.g., a recombinant therapeutic protein) from one or more othercomponents present in a liquid culture medium or a diluted liquidculture medium (e.g., culture medium proteins or one or more othercomponents (e.g., DNA, RNA, or other proteins) present in or secretedfrom a mammalian cell). Typically, capturing is performed using achromatography resin that binds a recombinant protein (e.g., through theuse of affinity chromatography). Non-limiting methods for capturing arecombinant protein from a liquid culture medium or diluted liquidculture medium are described herein and others are known in the art. Arecombinant protein can be captured from a liquid culture medium usingat least one chromatography column and/or chromatographic membrane(e.g., any of the chromatography columns and/or chromatographicmembranes described herein).

The term “purifying” means a step performed to isolate a recombinantprotein (e.g., a recombinant therapeutic protein) from one or more otherimpurities (e.g., bulk impurities) or components present in a fluidincluding a recombinant protein (e.g., liquid culture medium proteins orone or more other components (e.g., DNA, RNA, other proteins,endotoxins, viruses, etc.) present in or secreted from a mammaliancell). For example, purifying can be performed during or after aninitial capturing step. Purification can be performed using achromatography resin, membrane, or any other solid support that bindseither a recombinant protein or contaminants (e.g., through the use ofaffinity chromatography, hydrophobic interaction chromatography, anionor cation exchange chromatography, or molecular sieve chromatography). Arecombinant protein can be purified from a fluid including therecombinant protein using at least one chromatography column and/orchromatographic membrane (e.g., any of the chromatography columns orchromatographic membranes described herein).

The term “polishing” is a term of art and means a step performed toremove remaining trace or small amounts of contaminants or impuritiesfrom a fluid including a recombinant protein (e.g., a recombinanttherapeutic protein) that is close to a final desired purity. Forexample, polishing can be performed by passing a fluid including therecombinant protein through a chromatographic column(s) or membraneabsorber(s) that selectively binds to either the target recombinantprotein or small amounts of contaminants or impurities present in afluid including a recombinant protein. In such an example, theeluate/filtrate of the chromatographic column(s) or membrane absorber(s)includes the recombinant protein.

The term “filtering” means the removal of at least part of (e.g., atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99%) undesired biologicalcontaminants (e.g., a mammalian cell, bacteria, yeast cells, viruses, ormycobacteria) and/or particulate matter (e.g., precipitated proteins)from a liquid (e.g., a liquid culture medium or fluid present in any ofthe processes described herein).

The term “eluate/filtrate” is a term of art and means a fluid that isemitted from a chromatography column or chromatographic membrane thatincludes a detectable amount of a recombinant protein (e.g., arecombinant therapeutic protein).

The term “integrated process” means a process which is performed usingstructural elements that function cooperatively to achieve a specificresult (e.g., the purification of a recombinant protein from a liquidculture medium).

The term “continuous process” means a process which continuously feedsfluid through at least a part of the system. For example, a continuousprocess is a process which continuously feeds a liquid culture mediumincluding a recombinant protein from a bioreactor through a MCCS.Another example of a continuous process is a process which continuouslyfeeds a liquid culture medium including a recombinant protein from abioreactor through a first and second MCCS (MCCS1 and MCCS2). Additionalexamples include a process which continuously feeds a liquid culturemedium including a recombinant protein through a MCCS, a process thatcontinuously feeds a liquid culture medium including a recombinantprotein through a MCCS1 and a MCCS2, or a process that continuouslyfeeds a fluid including a recombinant protein through a MCCS2.

The term “closed process” is a term of art and means a process that isperformed such that components of the process (e.g., chromatographyresins and/or buffers) that come into contact with the recombinantprotein or liquids including the recombinant protein are notintentionally exposed to contaminating agents for a significant periodof time (e.g., not intentionally air-exposed for a significant period oftime).

The term “therapeutic protein drug substance” means a recombinantprotein (e.g., an immunoglobulin, protein fragment, engineered protein,or enzyme) that has been sufficiently purified or isolated fromcontaminating proteins, lipids, and nucleic acids (e.g., contaminatingproteins, lipids, and nucleic acids present in a liquid culture mediumor from a host cell (e.g., from a mammalian, yeast, or bacterial hostcell)) and biological contaminants (e.g., viral and bacterialcontaminants), and can be formulated into a pharmaceutical agent withoutany further substantial purification and/or decontamination step(s).

The term “multi-column chromatography system” or “MCCS” means a systemof a total of two or more interconnected or switching chromatographycolumns and/or chromatographic membranes. A non-limiting example of amulti-column chromatography system is a periodic counter currentchromatography system (PCC) including a total of two or moreinterconnected or switching chromatography columns and/orchromatographic membranes. Additional examples of multi-columnchromatography systems are described herein and are known in the art.

The term “substantially free” means a composition (e.g., a liquidculture medium) that is at least or about 90% free (e.g., at least orabout 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%free) of a specified substance (e.g., a mammalian cell or acontaminating protein, nucleic acid, carbohydrate, or lipid form amammalian cell).

The term “mammalian cell” means any cell from or derived from any mammal(e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow,or a rabbit). For example, a mammalian cell can be an immortalized cell.In some embodiments, the mammalian cell is a differentiated cell. Insome embodiments, the mammalian cell is an undifferentiated cell.Non-limiting examples of mammalian cells are described herein.Additional examples of mammalian cells are known in the art.

The term “culturing” or “cell culturing” means the maintenance orproliferation of a mammalian cell under a controlled set of physicalconditions.

The term “culture of mammalian cells” means a liquid culture mediumincluding a plurality of mammalian cells that is maintained orproliferated under a controlled set of physical conditions.

The term “liquid culture medium” means a fluid that includes sufficientnutrients to allow a cell (e.g., a mammalian cell) to grow orproliferate in vitro. For example, a liquid culture medium can includeone or more of: amino acids (e.g., 20 amino acids), a purine (e.g.,hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol,thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine,riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium,glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.In some embodiments, a liquid culture medium can include serum from amammal. In some embodiments, a liquid culture medium does not includeserum or another extract from a mammal (a defined liquid culturemedium). In some embodiments, a liquid culture medium can include tracemetals, a mammalian growth hormone, and/or a mammalian growth factor.Another example of liquid culture medium is minimal medium (e.g., amedium including only inorganic salts, a carbon source, and water).Non-limiting examples of liquid culture medium are described herein.Additional examples of liquid culture medium are known in the art andare commercially available. A liquid culture medium can include anydensity of mammalian cells. For example, as used herein, a volume ofliquid culture medium removed from a bioreactor can be substantiallyfree of mammalian cells.

The term “animal-derived component free liquid culture medium” means aliquid culture medium that does not include any components (e.g.,proteins or serum) derived from a mammal.

The term “serum-free liquid culture medium” means a liquid culturemedium that does not include a mammalian serum.

The term “serum-containing liquid culture medium” means a liquid culturemedium that includes a mammalian serum.

The term “chemically-defined liquid culture medium” is a term of art andmeans a liquid culture medium in which all of the chemical componentsare known. For example, a chemically-defined liquid culture medium doesnot include fetal bovine serum, bovine serum albumin, or human serumalbumin, as these preparations typically include a complex mix ofalbumins and lipids.

The term “protein-free liquid culture medium” means a liquid culturemedium that does not include any protein (e.g., any detectable protein).

The term “immunoglobulin” means a polypeptide including an amino acidsequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60,70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., avariable domain sequence, a framework sequence, and/or a constant domainsequence). The immunoglobulin may, for example, include at least 15amino acids of a light chain immunoglobulin, e.g., at least 15 aminoacids of a heavy chain immunoglobulin. The immunoglobulin may be anisolated antibody (e.g., an IgG, IgE, IgD, IgA, or IgM), e.g., asubclass of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). The immunoglobulinmay be an antibody fragment, e.g., a Fab fragment, a F(ab′)2 fragment,or an a scFv fragment. The immunoglobulin may also be a bi-specificantibody or a tri-specific antibody, or a dimer, trimer, or multimerantibody, or a diabody, an Affibody®, or a Nanobody®. The immunoglobulincan also be an engineered protein including at least one immunoglobulindomain (e.g., a fusion protein). Non-limiting examples ofimmunoglobulins are described herein and additional examples ofimmunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion ofa polypeptide sequence that is at least or about 4 amino acids, at leastor about 5 amino acids, at least or about 6 amino acids, at least orabout 7 amino acids, at least or about 8 amino acids, at least or about9 amino acids, at least or about 10 amino acids, at least or about 11amino acids, at least or about 12 amino acids, at least or about 13amino acids, at least or about 14 amino acids, at least or about 15amino acids, at least or about 16 amino acids, at least or about 17amino acids, at least or about 18 amino acids, at least or about 19amino acids, or at least or about 20 amino acids in length, or more than20 amino acids in length. A recombinant protein fragment can be producedusing any of the processes described herein.

The term “engineered protein” means a polypeptide that is not naturallyencoded by an endogenous nucleic acid present within an organism (e.g.,a mammal). Examples of engineered proteins include enzymes (e.g., withone or more amino acid substitutions, deletions, insertions, oradditions that result in an increase in stability and/or catalyticactivity of the engineered enzyme), fusion proteins, antibodies (e.g.,divalent antibodies, trivalent antibodies, or a diabody), andantigen-binding proteins that include at least one recombinantscaffolding sequence.

The term “secreted protein” or “secreted recombinant protein” means aprotein (e.g., a recombinant protein) that originally included at leastone secretion signal sequence when it is translated within a mammaliancell, and through, at least in part, enzymatic cleavage of the secretionsignal sequence in the mammalian cell, is secreted at least partiallyinto the extracellular space (e.g., a liquid culture medium). Skilledpractitioners will appreciate that a “secreted” protein need notdissociate entirely from the cell to be considered a secreted protein.

The term “perfusion bioreactor” means a bioreactor including a pluralityof cells (e.g., mammalian cells) in a first liquid culture medium,wherein the culturing of the cells present in the bioreactor includesperiodic or continuous removal of the first liquid culture medium and atthe same time or shortly thereafter adding substantially the same volumeof a second liquid culture medium to the bioreactor. In some examples,there is an incremental change (e.g., increase or decrease) in thevolume of the first liquid culture medium removed and added overincremental periods (e.g., an about 24-hour period, a period of betweenabout 1 minute and about 24-hours, or a period of greater than 24 hours)during the culturing period (e.g., the culture medium refeed rate on adaily basis). The fraction of media removed and replaced each day canvary depending on the particular cells being cultured, the initialseeding density, and the cell density at a particular time. “RV” or“reactor volume” means the volume of the culture medium present at thebeginning of the culturing process (e.g., the total volume of theculture medium present after seeding).

The term “fed-batch bioreactor” is a term of art and means a bioreactorincluding a plurality of cells (e.g., mammalian cells) in a first liquidculture medium, wherein the culturing of the cells present in thebioreactor includes the periodic or continuous addition of a secondliquid culture medium to the first liquid culture medium withoutsubstantial or significant removal of the first liquid culture medium orsecond liquid culture medium from the cell culture. The second liquidculture medium can be the same as the first liquid culture medium. Insome examples of fed-batch culture, the second liquid culture medium isa concentrated form of the first liquid culture medium. In some examplesof fed-batch culture, the second liquid culture medium is added as a drypowder.

The term “clarified liquid culture medium” means a liquid culture mediumobtained from a bacterial or yeast cell culture that is substantiallyfree (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free) ofbacteria or yeast cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is graph of the binding isotherms at t0 of untreated (virgin)multimodal resin with anionic exchange and hydrophobic groups (AE resin)and 25 kGy gamma-irradiated AE resin (without chromatography cycles).

FIG. 2 is a graph of the percentage normalized binding capacity ofvirgin (untreated) AE resin, 15 kGy gamma-irradiated AE resin, and 25kGy gamma-irradiated AE resin over multiple cycles of columnchromatography, when a buffer of 700 mM arginine, 100 mM acetate (pH3.0), followed by a solution of 1 N NaOH is used to wash each resin(after elution of the recombinant protein) in each cycle. The percentagebinding capacity of each resin is normalized to the binding capacity ofvirgin (untreated) AE resin at time 0 (t0), which was also found to bevery similar to gamma-irradiated resin at t0 (27±2 mg/mL).

FIG. 3 is a graph showing the mean rate of drop in binding capacity overmultiple cycles of column chromatography for virgin (untreated) AEresin, 15 kGy gamma-irradiated AE resin, and 25 kGy gamma-irradiated AE,when a buffer of 700 mM arginine, 100 mM acetate (pH 3.0), followed by asolution of 1 N NaOH is used to wash each resin (after elution of therecombinant protein) in each cycle.

FIG. 4 is a graph of the percentage normalized binding capacity ofvirgin (untreated) AE resin over multiple cycles of columnchromatography using a single chromatography column including virgin(untreated) AE resin washed with 700 mM arginine, 100 mM acetate (pH3.0), followed by a solution of 1 N NaOH after elution of therecombinant protein in each cycle (low pH arginine virgin), or using asingle chromatography column including 25 kGy gamma-irradiated AE resinwashed after elution of the recombinant protein (in each cycle) with 8 Murea, 1 M NaCl, 0.1 M citric acid, pH 2.5 (low pH urea); 6 M guanidineHCl (pH 2.5) (low pH guanidine); 0.5% Triton-X 100 in 0.1 M acetic acid(pH 2.5) followed by 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol(pH 2.5) (low pH Triton); 700 mM arginine, 100 mM acetate (pH 3.0) (lowpH arginine); or 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol (pH2.5) (low pH organic), each followed by a solution of 1 N NaOH. Thepercentage binding capacity of each resin over each cycle was normalizedto the binding capacity of virgin (untreated) AE resin at time 0 (t0),which was also found to be very similar to gamma-irradiated resin at t0(27±2 mg/mL).

FIG. 5 is a representative chromatograph profile from a multi-columnchromatography (MCC) run showing the eluate absorbance at 280 nm overmultiple cycles of column chromatography performed using threechromatography columns including 25 kGy irradiated AE resin, when 8 Murea, 1 M NaCl, 0.1 M citric acid, pH 2.5, followed by a solution of 1 NNaOH is used to wash each resin (after elution of recombinant protein)in each cycle. The peaks representing the bound protein released fromthe AE resin during exposure to the 8M urea, 1 M NaCl, 0.1 M citricacid, pH 2.5 denaturing buffer in three different cycles are indicatedwith an arrow. The chromatograph shows the UV trace for all threechromatography columns used to perform the MCC run.

FIG. 6 is a graph of the percentage recovered recombinant protein boundto the chromatography resin per cycle over multiple cycles of columnchromatography using a single chromatography column including virgin(untreated) AE resin washed with 700 mM arginine, 100 mM acetate (pH3.0), followed by a solution of 1 N NaOH after elution of therecombinant protein in each cycle (low pH arginine virgin), or using asingle chromatography column including 25 kGy gamma-irradiated AE resinwashed after elution of the recombinant protein (in each cycle) with 8 Murea, 1 M NaCl, 0.1 M citric acid, pH 2.5 (low pH urea); 6 M guanidineHCl (pH 2.5) (low pH guanidine); 0.5% Triton-X 100 in 0.1 M acetic acid(pH 2.5) followed by 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol(pH 2.5) (low pH Triton); or 0.7 M acetic acid, 20% ethanol, 50%ethylene glycol (pH 2.5) (low pH organic); each followed by a solutionof 1 N NaOH.

FIG. 7 is a graph of the host cell protein (ng/mg) present in eluateover multiple cycles of column chromatography using a singlechromatography column including virgin (untreated) AE resin washed with700 mM arginine, 100 mM acetate (pH 3.0), followed by a solution of 1 NNaOH after elution of the recombinant protein in each cycle (low pHarginine virgin), or using a single chromatography column including 25kGy gamma-irradiated AE resin washed after elution of the recombinantprotein (in each cycle) with 8 M urea, 1 M NaCl, 0.1 M citric acid, pH2.5 (low pH urea); 6 M guanidine HCl (pH 2.5) (low pH guanidine); 0.5%Triton-X 100 in 0.1 M acetic acid (pH 2.5) followed by 0.7 M aceticacid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pH Triton); or 0.7M acetic acid, 20% ethanol, 50% ethylene glycol (pH 2.5) (low pHorganic); each followed by a solution of 1 N NaOH.

FIG. 8 is list of lysosomal storage diseases and the recombinanttherapeutic enzyme that can be used to treat each disease.

DETAILED DESCRIPTION

Provided herein are methods of performing chromatography withgamma-irradiated chromatography resin that include providing achromatography column including a gamma-irradiated chromatography resinand performing a first cycle of chromatography through the column, wherethe cycle includes exposing the chromatography resin to a denaturingbuffer. Also provided are processes integrated, closed or substantiallyclosed, and continuous processes for manufacturing of a recombinantprotein that include the use of at least one chromatography columnincluding gamma-irradiated chromatography resin, where thegamma-irradiated chromatography resin is exposed to denaturing bufferduring each cycle in the process, and reduced bioburden buffer is usedin the process. Non-limiting aspects of these methods and processes aredescribed below. As can be appreciated in the art, the various aspectsdescribed below can be used in any combination without limitation.

Gamma-Irradiated Chromatography Resin

A wide variety of different types of chromatography resin known in theart (or combinations thereof) can be exposed to gamma-irradiation usingmethods known in the art. For example, an isotope such as Cobalt-60 orCaesium-137 is used as the source of gamma-rays. The chromatographyresin that is exposed to gamma-irradiation can be present in a packedchromatography column. In other examples, the chromatography resin thatis exposed to gamma-irradiation is present in a sealed container (e.g.,a slurry in a sealed container).

The chromatography resin can be exposed to gamma-irradiation at atemperature of about between about −25° C. and about 0° C., inclusive,or between about 0° C. and about 25° C., inclusive. The chromagraphyresin can be exposed to a dose of gamma-irradiation of between about 0.1kGy to about 100 kGy, between about 1 kGy to about 100 kGy, betweenabout 1 kGy to about 90 kGy, between about 1 kGy to about 80 kGy,between about 1 kGy to about 70 kGy, between about 1 kGy to about 65kGy, between about 5 kGy to about 65 kGy, between about 10 kGy to about60 kGy, between about 10 kGy to about 55 kGy, between about 10 kGy toabout 50 kGy, between about 10 kGy to about 45 kGy, between about 10 kGyto about 40 kGy, between about 10 kGy to about 35 kGy, between about 10kGy to about 30 kGy, between about 15 kGy to about 50 kGy, between about15 kGy to about 45 kGy, between about 15 kGy to about 40 kGy, betweenabout 15 kGy to about 35 kGy, or between about 20 kGy to about 30 kGy.

The gamma-irradiated chromatography resin can be an anionic exchangechromatography resin, cationic exchange chromatography resin, sizeexclusion chromatography resin, hydrophobic interaction chromatographyresin, affinity chromatography resin, or any combination thereof.Non-limiting examples of affinity chromatography resin include resinswith a peptide ligand, a protein ligand (e.g., protein A or protein G),an aptamer ligand, a substrate ligand, a product ligand, a metal ligand,and a cofactor ligand. A gamma-irradiated chromatography resin can be abimodal chromatography resin (e.g., with the features of anionicexchange and hydrophobic interaction chromatography resin).

Chromatography Columns Including Gamma-Irradiated Chromatography Resin

The methods described herein include the use of a chromatography columnincluding gamma-irradiated chromatography resin and the processesdescribed herein include the use of one or two MCCSs that include atleast one chromatography column including a gamma-irradiatedchromatography resin. The gamma-irradiated chromatography resin can beany type of resin described herein (or any type of chromatography resinknown in the art). The gamma-irradiated chromatography resin can beprepared using any of the methods described herein or known in the art.

Such chromatography columns can be produced by packing a chromatographycolumn with an untreated chromatography resin(s), and exposing thepacked column to gamma-irradiation (e.g., using any of the exposures andconditions described herein). In other examples, chromatography columnsincluding a gamma-irradiated resin(s) can be produced by exposing thechromatography resin to gamma-irradiation (e.g., chromatography resinprovided in a container) and packing a chromatography column with thegamma-irradiated chromatography resin. In such methods, thechromatography resin that is exposed to gamma-irradiation can be presentas a slurry in the container, and the chromatography column is packed ina reduced bioburden hood. In other methods, the chromatography resin canbe exposed to gamma-irradiation as a solid mixture in the container, anda slurry of the gamma-irradiated chromatography resin can be preparedusing a reduced bioburden buffer (e.g., prepared in a reduced bioburdenhood), and the resulting slurry used to pack a chromatography column ina reduced bioburden hood. In some of these examples, the chromatographycolumn, prior to packing, can be treated to reduce the bioburden (e.g.,autoclaved, gamma-irradiated, or exposure to ethylene oxide).

The chromatography column including gamma-irradiated chromatographyresin can have a sterility assurance level (SAL) of between about 1×10⁻³and about 1×10⁻¹², between about 1×10⁻⁴ and about 1×10⁻¹², between1×10⁻⁵ and about 1×10⁻¹¹, between about 1×10⁻⁵ and about 1×10⁻¹⁰,between about 1×10⁻⁵ and about 1×10⁻⁹, between about 1×10⁻⁶ and about1×10⁻⁹, or between about 1×10⁻⁶ and about 1×10⁻⁸, inclusive.

Reduced Bioburden Buffers

The methods and processes described herein can be performed using one ormore reduced bioburden buffers. As can be appreciated in the art, areduced bioburden buffer can be any type of buffer used in a cycle ofchromatography (e.g., a buffer used in any of the steps in a cycle ofchromatography or in any of the unit operations described herein).Exemplary methods for reducing the bioburden of a buffer includefiltration (0.2 μm-pore size filtration), autoclaving, andgamma-irradiation. Additional methods for reducing the bioburden of abuffer are known in the art. A reduced bioburden buffer can have asterility assurance level of between about 1×10⁻³ and about 1×10⁻¹²,between about 1×10⁻⁴ and about 1×10⁻¹², between 1×10⁻⁵ and about1×10⁻¹¹, between about 1×10⁻⁵ and about 1×10⁻¹⁰, between about 1×10⁻⁵and about 1×10⁻⁹, between about 1×10⁻⁶ and about 1×10⁻⁹, or betweenabout 1×10⁻⁶ and about 1×10⁻⁸, inclusive.

Denaturing Buffers

The methods and processes described herein include the use of adenaturing buffer. Denaturing buffers include a sufficient amount of oneor more chemical agents (e.g., detergent(s), reductant(s), acid(s),chaotropic agent(s), organic solvent(s), or crosslinking agent(s), orany combination thereof) that result in the denaturation of a protein.Non-limiting exemplary detergents that can be included in a denaturingbuffer include Triton X-100, sodium dodecyl sulfate, and ethyl trimethylammonium bromide. Non-limiting examples of organic solvents that can beincluded in a denaturing buffer include ethanol, butanol, phenol,propanol, and methanol. Non-limiting examples of reductants that can beincluded in a denaturing buffer include 2-mercaptoethanol,dithiothreitol, and tris(2-carboxyethyl)phosphine. Non-limiting examplesof acids that can be included in denaturing buffers include acetic acid,thichloroacetic acid, and sulfosalicylic acid. Non-limiting examples ofchaotropic agents that can be included in a denaturing buffer includeurea (e.g., 6 to 9 M urea), thiourea, guanidium chloride (e.g., 5 to 7 Mguanidium chloride), lithium perchlorate (e.g., 4 to 7 M lithiumperchlorate), or lithium acetate. Non-limiting examples of cross-linkingagents that can be included in a denaturing buffer include formaldehydeand glutaraldehyde.

Non-limiting examples of denaturing buffers include: 8 M urea, 1 M NaCl,0.1 M citric acid, pH 2.5 (e.g., followed by 1 N NaOH); 6 M guanidineHCl, pH 2.5 (e.g., followed by 1 N NaOH or 1 M NaOH plus 1 M NaCl); and0.5% Triton-X 100 in 0.1 M acetic acid, pH 2.5 (e.g., followed by 0.7 Macetic acid, 20% ethanol, 50% ethylene glycol, pH 2.5, followed by 1 NNaOH).

Recombinant Therapeutic Proteins

A recombinant protein as described herein can be a recombinanttherapeutic protein. Non-limiting examples of recombinant therapeuticproteins that can be produced by the methods provided herein includeimmunoglobulins (including light and heavy chain immunoglobulins,antibodies, or antibody fragments (e.g., any of the antibody fragmentsdescribed herein), enzymes (e.g., a galactosidase (e.g., analpha-galactosidase), Myozyme®, or Cerezyme®), proteins (e.g., humanerythropoietin, tumor necrosis factor (TNF), or an interferon alpha orbeta), or immunogenic or antigenic proteins or protein fragments (e.g.,proteins for use in a vaccine). Non-limiting examples of recombinanttherapeutic enzymes that can be used to treat a variety of lysosomalstorage diseases are shown in FIG. 8. The recombinant therapeuticprotein can be an engineered antigen-binding polypeptide that includesat least one multifunctional recombinant protein scaffold (see, e.g.,the recombinant antigen-binding proteins described in Gebauer et al.,Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent ApplicationPublication No. 2012/0164066 (herein incorporated by reference in itsentirety)). Non-limiting examples of recombinant therapeutic proteinsthat are antibodies include: panitumumab, omalizumab, abagovomab,abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab,alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab,anatumomab, apolizumab, atinumab, tocilizumab, basilizimab, bectumomab,belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab,densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab,etaracizumab, golimumab, infliximab, natalizumab, palivizumab,panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, andtrastuzumab. Additional examples of recombinant therapeutic antibodiesthat can be produced by the methods described herein are known in theart. Additional non-limiting examples of recombinant therapeuticproteins that can be produced/purified by the present methods include:alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa,antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetinalfa, tenecteplase, etanercept, coagulation factor IX, folliclestimulating hormone, interferon beta-1a, imiglucerase, dornase alfa,epoetin alfa, and alteplase.

A secreted, soluble recombinant therapeutic protein can be recoveredfrom the liquid culture medium (e.g., a first and/or second liquidculture medium) by removing or otherwise physically separating theliquid culture medium from the cells (e.g., mammalian cells). A varietyof different methods for removing liquid culture medium from cells(e.g., mammalian cells) are known in the art, including, for example,centrifugation, filtration, pipetting, and/or aspiration. The secretedrecombinant therapeutic protein can then be recovered and furtherpurified from the liquid culture medium using a variety of biochemicaltechniques including various types of chromatography (e.g., affinitychromatography, molecular sieve chromatography, cation exchangechromatography, anion exchange chromatography, or hydrophobicinteraction chromatography, or any combination thereof) and/orfiltration (e.g., molecular weight cut-off filtration).

Cycle of Chromatography

As is well-known in the art, the steps in a cycle of chromatography candiffer depending on the chromatography resin, the buffers used toperform each step in the cycle, and the biophysical characteristics ofthe target recombinant protein (e.g., recombinant therapeutic protein).For example, an affinity chromatography column can include the steps ofloading an affinity chromatography column with a fluid including thetarget recombinant protein, washing the column to remove unwantedbiological material (e.g., contaminating proteins and/or smallmolecules), eluting the target recombinant protein bound to the column,and re-equilibrating the column. A cycle of chromatography using acationic and/or anionic exchange chromatography column, where the targetrecombinant protein binds to the chromatography resin in the loadingstep, can include the steps of loading the column with a fluid includingthe target protein, washing the column to remove unwanted biologicalmaterial, eluting the target recombinant protein bound to the column,and re-equilibrating the column. In other examples, a cycle ofchromatography using a cationic and/or anionic exchange chromatographycolumn, where unwanted biological material binds to the chromatographyresin during the loading step, while the target recombinant protein doesnot, can include the steps of loading the column with a fluid includingthe target protein, collecting the target recombinant protein in theflow-through, and reequilibrating the column. As is well-known in theart, any of the single steps in a chromatography cycle can include asingle buffer or multiple buffers (e.g., two or more buffers), and oneor more of any of the single steps in a chromatography cycle can includea buffer gradient. Any of the combination of various well-known aspectsof a single cycle of chromatography can be used in these methods in anycombination, e.g., different chromatography resin(s), flow-rate(s),buffer(s), void volume(s) of the column, bed volume(s) of the column,volume(s) of buffer used in each step, volume(s) of the fluid includingthe target protein, and the number and types of buffer(s) used in eachstep.

Methods of Performing Chromatography with Gamma-Sterilized Resin

Provided herein are methods of performing chromatography withgamma-irradiated chromatography resin. These methods include providing achromatography column including a gamma-irradiated chromatography resin,performing a first cycle of chromatography through the column, whereinthe cycle includes exposing the chromatography resin to a denaturingbuffer, and performing at least one additional cycle of chromatographythrough the column. The gamma-irradiated chromatography resin can be anytype of chromatography resin and/or can be any of the gamma-irradiatedchromatography resins described herein or known in the art. Thechromatography column can be any of the chromatography columns includinga gamma-irradiated chromatography resin described herein or known in theart. The recombinant protein can be a recombinant therapeutic protein(e.g., any of the recombinant therapeutic proteins described herein orknown in the art).

In some examples, the column is part of a multi-column chromatographysystem (MCCS), e.g., can be part of a periodic counter currentchromatography system (PCCS).

The denaturing buffer can be any of the exemplary denaturing buffersdescribed herein or known in the art. For example, the denaturing buffercan include one or more of urea, guanidine hydrochloride, and Triton™X-100. In some examples, the chromatography resin is exposed to thedenaturing buffer for a period of between at least 1 minute to about 2hours (e.g., between 1 minute and about 1.5 hours, between about 1minute and about 1.0 hour, between about 1 minute and about 55 minutes,between about 1 minute and about 50 minutes, between about 1 minute andabout 45 minutes, between about 1 minute and about 40 minutes, betweenabout 1 minute and about 40 minutes, between about 1 minute and about 35minutes, between about 1 minute and about 30 minutes, between about 1minute and about 25 minutes, between about 1 minute and about 20minutes, between about 1 minute and about 15 minutes, or between about 1minute and about 10 minutes). In some examples, exposing thechromatography resin to denaturing buffer include passing between about0.5× bed volume to about 10× bed volume (e.g., between about 0.5× bedvolume to about 9.0× bed volume, between about 0.5× bed volume to about8.0× bed volume, between about 0.5× bed volume to about 7.0× bed volume,between about 0.5× bed volume to about 6.0× bed volume, between about0.5× bed volume to about 5.0× bed volume, between about 0.5× bed volumeto about 4.0× bed volume, between about 0.5× bed volume to about 3.5×bed volume, between about 0.5× bed volume to about 3.0× bed volume,between about 0.5× bed volume to about 2.5× bed volume, between about0.5× bed volume to about 2.0× bed volume, or between about 0.5× bedvolume to about 1.5× bed volume) of denaturing buffer through thechromatography column. In some examples, the first cycle ofchromatography can include exposing the chromatography resin to washbuffer comprising about 0.5 M to about 1.5 M sodium hydroxide followingexposure to the denaturing buffer.

The first cycle of chromatography performed through the column can beany cycle of chromatography described herein or known in the art thatincludes exposing the chromatography resin to a denaturing buffer. Theat least one additional cycle of chromatography performed through thecolumn can be any cycle of chromatography described herein or known inthe art. For example, the first cycle of chromatography and the at leastone additional cycle of chromatography can include the steps of:capturing the recombinant protein by exposing the chromatography resinwith a liquid including a recombinant protein; washing thechromatography resin by exposing the chromatography resin with a washbuffer, eluting the recombinant protein by exposing the chromatographyresin with an elution buffer; and regenerating the chromatography resinby exposing the chromatography resin to the denaturing buffer. In someexamples, the liquid including the recombinant protein is a liquidculture medium (e.g., a liquid culture medium collected from a perfusionor batch culture).

The first chromatography cycle and the at least one additionalchromatography cycle can be performed using a closed and integratedsystem (e.g., any of the exemplary closed and integrated systemsdescribed herein or known in the art). For example, the firstchromatography cycle and the at least one additional chromatographycycle can be performed using a closed and integrated system, where thebuffer is reduced bioburden buffer (e.g., all the buffers used in thefirst and at least one additional cycles). As is well-known in the art,reduced bioburden buffer can be produced using a variety of differentmethods (e.g., prepared by filtration, by autoclaving, or heattreatment).

The at least one additional cycle of chromatography can be at two ormore (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 ormore, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 ormore, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, or 100or more) cycles of additional chromatography. In some examples, the atleast one additional cycle of chromatography is performed continuouslyover a period of at least 3 days (e.g., at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 8 days, at least 9days, at least 10 days, at least 11 days, at least 12 days, at least 13days, at least 14 days, at least 15 days, at least 16 days, at least 17days, at least 18 days, at least 19 days, at least 20 days, at least 21days, at least 22 days, at least 23 days, at least 24 days, at least 25days, at least 30 days, at least 35 days, at least 40 days, at least 45days, at least 50 days, at least 55 days, at least 60 days, at least 65days, at least 70 days, at least 75 days, at least 80 days, at least 85days, at least 90 days, at least 95 days, or at least 100 days).

Integrated, Closed or Substantially Closed, and Continuous Processes forManufacturing of a Recombinant Protein

Provided herein are integrated, closed or substantially closed, andcontinuous processes for manufacturing a purified recombinant protein(e.g., a recombinant therapeutic protein). These processes includeproviding a liquid culture medium including a recombinant protein (e.g.,a recombinant therapeutic protein) that is substantially free of cells.

Some processes include continuously feeding the liquid culture mediuminto a multi-column chromatography system (MCCS) that includes at leastone chromatography column including gamma-sterilized resin, where thechromatography resin is exposed during each cycle to denaturing buffer(e.g., exposed to any of the denaturing buffers described herein orknown in the art for any of the durations described herein). Theseprocesses utilize reduced bioburden buffer, are integrated, and runcontinuously from the liquid culture medium to an eluate from the MCCSthat is the purified recombinant protein (e.g., a therapeutic proteindrug substance).

Some processes include continuously feeding the liquid culture mediuminto a first MCCS (MCCS1), capturing the recombinant protein from theliquid culture medium using the MCCS1, producing an eluate from theMCCS1 that includes the recombinant protein and continuously feeding theeluate into a second MCCS (MCCS2), and continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, where at least one column in the MCCS1 and/or theMCCS2 is a chromatography column that includes gamma-irradiatedchromatography resin and the chromatography resin is exposed todenaturing buffer during each cycle in the process (e.g., exposed to anyof the denaturing buffers described herein or known in the art for anyof the durations described herein). These processes utilize reducedbioburden buffer, are integrated, and run continuously from the liquidculture medium to the purified recombinant protein.

In some examples, each of the chromatography columns used in the MCCS,MCCS1, and/or MCCS2 includes a gamma-irradiated chromatography resin.Some embodiments further include a step of formulating the purifiedrecombinant protein into a pharmaceutical composition.

The processes described herein provide continuous and time-efficientproduction of a purified recombinant protein from a liquid culturemedium including the recombinant protein. For example, the elapsed timebetween feeding a liquid culture medium including a therapeutic proteininto the MCCS or MCCS1 and eluting the recombinant protein from the MCCSor MCCS2, respectively, can be, e.g., between about 4 hours and about 48hours, inclusive, e.g., between about 4 hours and about 40 hours,between about 4 hours and about 35 hours, between about 4 hours andabout 30 hours, between about 4 hours and about 28 hours, between about4 hours and about 26 hours, between about 4 hours and about 24 hours,between about 4 hours and about 22 hours, between about 4 hours andabout 20 hours, between about 4 hours and about 18 hours, between about4 hours and about 16 hours, between about 4 hours and about 14 hours,between about 4 hours and about 12 hours, between about 6 hours andabout 12 hours, between about 8 hours and about 12 hours, between about6 hours and about 20 hours, between about 6 hours and about 18 hours,between about 6 hours and about 14 hours, between about 8 hours andabout 16 hours, between about 8 hours and about 14 hours, between about8 hours and about 12 hours, between about 10 hours and 20 hours, betweenabout 10 hours and 18 hours, between about 10 hours and 16 hours,between about 10 hours and 14 hours, between about 12 hours and about 14hours, between about 10 hours and about 40 hours, between about 10 hoursand about 35 hours, between about 10 hours and about 30 hours, betweenabout 10 hours and about 25 hours, between about 15 hours and about 40hours, between about 15 hours and about 35 hours, between about 15 hoursand about 30 hours, between about 20 hours and about 40 hours, betweenabout 20 hours and about 35 hours, or between about 20 hours and about30 hours, inclusive. In other examples, the elapsed time between feedingthe liquid culture medium including the recombinant protein into theMCCS or MCCS1 and eluting the recombinant protein from the MCCS orMCCS2, respectively, is, e.g., greater than about 4 hours and less thanabout 40 hours, inclusive, e.g., greater than about 4 hours and lessthan about 39 hours, about 38 hours, about 37 hours, about 36 hours,about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31hours, about 30 hours, about 29 hours, about 28 hours, about 27 hours,about 26 hours, about 25 hours, about 24 hours, about 23 hours, about 22hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours,about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5hours, inclusive.

Non-limiting aspects of the MCCSs that can be used in any of theseprocesses (MCCS, MCCS1, and/or MCCS2) are described in U.S. ProvisionalPatent Application Ser. Nos. 61/775,060 and 61/856,390 (eachincorporated herein by reference).

Some exemplary processes do not utilize a holding step (e.g., do not usea reservoir (e.g., break tank) in the entire process). Others may use amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process. Any of the processes described herein can utilize amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process, where each break tank only holds a recombinant proteinfor a total time period of, e.g., between about 5 minutes and less thanabout 6 hours, inclusive, e.g., between about 5 minutes and about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 30 minutes, inclusive.

Some processes utilize one, two, three, four, five, or six reservoir(s)(e.g., break tank(s)) and can have a capacity that is, e.g., between 1mL and about 300 mL, inclusive, e.g., between 1 mL and about 280 mL,about 260 mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL,about 160 mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL,about 60 mL, about 40 mL, about 20 mL, or about 10 mL (inclusive). Anyreservoir(s) (e.g., break tank(s)) used (in any of the processesdescribed herein) to hold fluid before it is fed into the MCCS or MCCS1can have a capacity that is, e.g., between 1 mL and about 100%,inclusive, e.g., between 1 mL and about 90%, about 80%, about 70%, about60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%,inclusive, of the loading volume of the first column of the MCCS orMCCS1. A reservoir(s) (e.g., break tanks(s)) can be used to hold eluatefrom MCCS1 before it enters into the MCCS2 and can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, e.g., between 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the MCCS2.

Various additional aspects of these processes are described in detailbelow and can be used in any combination in the processes providedherein without limitation. Exemplary aspects of the provided processesare described below; however, one skilled in the art will appreciatethat additional steps can be added to the processes described herein andother materials can be used to perform any of the steps of the processesdescribed herein.

Liquid Culture Medium

Liquid culture medium that includes a recombinant protein (e.g.,recombinant therapeutic protein) that is substantially free of cells canbe derived from any source. For example, the liquid culture medium canbe obtained from a recombinant cell culture (e.g., a recombinantbacterial, yeast, or mammalian cell culture). The liquid culture mediumcan be obtained from a fed-batch cell (e.g., mammalian cell) culture(e.g., a fed-batch bioreactor including a culture of mammalian cellsthat secrete the recombinant protein) or a perfusion cell (e.g.,mammalian cell) culture (e.g., a perfusion bioreactor including aculture of mammalian cells that secrete the recombinant protein). Theliquid culture medium can also be a clarified liquid culture medium froma culture of bacterial or yeast cells that secrete the recombinantprotein.

Liquid culture medium obtained from a recombinant cell culture can befiltered or clarified to obtain a liquid culture medium that issubstantially free of cells and/or viruses. Methods for filtering orclarifying a liquid culture medium in order to remove cells are known inthe art (e.g., 0.2-μm filtration and filtration using an AlternatingTangential Flow (ATF™) system). Recombinant cells can also be removedfrom liquid culture medium using centrifugation and removing thesupernatant that is liquid culture medium that is substantially free ofcells, or by allowing the cells to settle to the gravitational bottom ofa container (e.g., bioreactor) including the liquid culture medium, andremoving the liquid culture medium (the liquid culture medium that issubstantially free of cells) that is distant from the settledrecombinant cells.

The liquid culture medium can be obtained from a culture of recombinantcells (e.g., recombinant bacteria, yeast, or mammalian cells) producingany of the recombinant proteins (e.g., recombinant therapeutic proteins)described herein or known in the art. Some examples of any of theprocesses described herein can further include a step of culturingrecombinant cells (e.g., recombinant bacteria, yeast, or mammaliancells) that produce the recombinant protein (e.g., recombinanttherapeutic protein).

The liquid culture medium can be any of the types of liquid culturemedium described herein or known in the art. For example, the liquidculture medium can be selected from the group of: animal-derivedcomponent-free liquid culture medium, serum-free liquid culture medium,serum-containing liquid culture medium, chemically-defined liquidculture medium, and protein-free liquid culture medium. In any of theprocesses described herein, a liquid culture medium obtained from aculture can be diluted by addition of a second fluid (e.g., a buffer)before it is fed into the MCCS or MCCS1.

The liquid culture medium including a recombinant protein that issubstantially free of cells can be stored (e.g., at a temperature belowabout 15° C. (e.g., below about 10° C., below about 4° C., below about0° C., below about −20° C., below about −50° C., below about −70 C.°, orbelow about −80° C.) for at least 1 day (e.g., at least about 2 days, atleast about 5 days, at least about 10 days, at least about 15 days, atleast about 20 days, or at least about 30 days) prior to feeding theliquid culture medium into the MCCS or MCCS1. Alternatively, in someexamples the liquid culture medium is fed into the MCCS or MCCS1directly from a bioreactor (e.g., fed into the MCCS or MCCS1 directlyfrom the bioreactor after a filtering or clarification step).

Multi-Column Chromatography Systems

The processes described herein include the use of a MCCS or two or more(e.g., two, three, four, five, or six) multi-column chromatographysystems (MCCSs) (e.g., an MCCS1 and MCCS2). A MCCS can include two ormore chromatography columns, two or more chromatographic membranes, or acombination of at least one chromatography column and at least onechromatographic membrane. In non-limiting examples, a MCCS (e.g., MCCS,MCCS1, and/or MCCS2 in any of the processes herein) can include fourchromatographic columns, three chromatographic columns and achromatographic membrane, three chromatographic columns, twochromatographic columns, two chromatographic membranes, and twochromatographic columns and one chromatographic membrane. Additionalexamples of combinations of chromatography columns and/orchromatographic membranes can be envisioned for use in an MCCS (e.g.,MCCS, MCCS1, and/or MCCS2 in any of the processes described herein) byone skilled in the art without limitation. The individual chromatographycolumns and/or chromatographic membranes present in a MCCS can beidentical (e.g., have the same shape, volume, resin, capture mechanism,and unit operation), or can be different (e.g., have one or more of adifferent shape, volume, resin, capture mechanism, and unit operation).The individual chromatography column(s) and/or chromatographicmembrane(s) present in a MCCS (e.g., MCCS, MCCS1, and/or MCCS2 in any ofthe processes described herein) can perform the same unit operation(e.g., the unit operation of capturing, purifying, or polishing) ordifferent unit operations (e.g., different unit operations selectedfrom, e.g., the group of capturing, purifying, polishing, inactivatingviruses, adjusting the ionic concentration and/or pH of a fluidincluding the recombinant protein, and filtering). For example, inexamples of the processes described herein, at least one chromatographycolumn and/or chromatographic membrane in the MCCS or MCCS1 performs theunit operation of capturing the recombinant protein.

The one or more chromatography column(s) that can be present in an MCCS(e.g., present in the MCCS, MCCS1, and/or MCCS2) can have a resin volumeof, e.g., between about 1 mL and about 2 mL, about 5 mL, about 10 mL,about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL,about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about95 mL, or about 100 mL, inclusive. The one or more chromatographycolumn(s) that can be present in an MCCS (e.g., present in the MCCS,MCCS1, and/or MCCS2) can have a resin volume of between about 2 mL toabout 100 mL, between about 2 mL and about 90 mL, between about 2 mL andabout 80 mL, between about 2 mL and about 70 mL, between about 2 mL andabout 60 mL, between about 2 mL and about 50 mL, between about 5 mL andabout 50 mL, between about 2 mL and about 45 mL, between about 5 mL andabout 45 mL, between about 2 mL and about 40 mL, between about 5 mL andabout 40 mL, between about 2 mL and about 35 mL, between about 5 mL andabout 35 mL, between about 2 mL and about 30 mL, between about 5 mL andabout 30 mL, between about 2 mL and about 25 mL, between about 5 mL andabout 25 mL, between about 15 mL and about 60 mL, between about 10 mLand about 60 mL, between about 10 mL and about 50 mL, and between about15 mL and about 50 mL. The one or more chromatography column(s) in anMCCS (e.g., the MCCS, MCCS1, and/or MCCS2) used in any of the processesdescribed herein can have the substantially the same resin volume or canhave different resin volumes. The flow rate used for the one or morechromatography column(s) in an MCCS (e.g., the MCCS, MCCS1, and/orMCCS2) can be, e.g., between about 0.2 mL/minute to about 25 mL/minute(e.g., between about 0.2 mL/minute to about 20 mL/minute, between about0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minute toabout 15 mL/minute, between about 0.5 mL/minute to about 15 mL/minute,between about 0.5 mL/minute to about 10 mL/minute, between about 0.5 mLminute and about 14 mL/minute, between about 1.0 mL/minute and about25.0 mL/minute, between about 1.0 mL/minute and about 15.0 mL/minute).

The one or more chromatography column (s) in an MCCS (e.g., MCCS, MCCS1,and/or MCCS2) can have substantially the same shape or can havesubstantially different shapes. For example, the one or morechromatography column(s) in an MCCS (e.g., in the MCCS, MCCS1, and/orMCCS2) can have substantially the shape of a circular cylinder or canhave substantially the same shape of an oval cylinder.

The one or more chromatographic membrane(s) that can be present in anMCCS (e.g., present in the MCCS, MCCS1, and/or MCCS2) can have a bedvolume of, e.g., between about 1 mL to about 500 mL (e.g., between about1 mL to about 475 mL, between about 1 mL to about 450 mL, between about1 mL to about 425 mL, between about 1 mL to about 400 mL, between about1 mL to about 375 mL, between about 1 mL to about 350 mL, between about1 mL to about 325 mL, between about 1 mL to about 300 mL, between about1 mL to about 275 mL, between about 1 mL to about 250 mL, between about1 mL to about 225 mL, between about 1 mL to about 200 mL, between about1 mL to about 175 mL, between about 1 mL to about 150 mL, between about1 mL to about 125 mL, between about 1 mL to about 100 mL, between about2 mL to about 100 mL, between about 5 mL to about 100 mL, between about1 mL to about 80 mL, between about 2 mL to about 80 mL, between about 5mL to about 80 mL, between about 1 mL to about 60 mL, between about 2 mLto about 60 mL, between about 5 mL to about 60 mL, between about 1 mL toabout 40 mL, between about 2 mL to about 40 mL, between about 5 mL toabout 40 mL, between about 1 mL to about 30 mL, between about 2 mL toabout 30 mL, between about 5 mL to about 30 mL, between about 1 mL andabout 25 mL, between about 2 mL and about 25 mL, between about 1 mL andabout 20 mL, between about 2 mL and about 20 mL, between about 1 mL andabout 15 mL, between about 2 mL and about 15 mL, between about 1 mL andabout 10 mL, or between about 2 mL and about 10 mL).

One or more (e.g., three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, ortwenty-four) different types of reduced bioburden buffer can be employedduring the use of the MCCS, MCCS1, and/or MCCS2 in any of the processesdescribed herein. As is known in the art, the one or more types ofreduced bioburden buffer used in the MCCS, MCCS1, and/or MCCS2 in theprocesses described herein will depend on the resin present in thechromatography column(s) and/or the chromatographic membrane(s) of theMCCS, MCCS1, and/or MCCS2, the biophysical properties of the recombinantprotein, and unit operation (e.g., any of the exemplary unit operationsdescribed herein) performed by the specific chromatography column(s)and/or chromatography membranes of the MCCS, MCCS1, and/or MCCS2. Thevolume and type of buffer employed during the use of the MCCS, MCCS1,and/or MCCS2 in any of the processes described herein can also bedetermined by one skilled in the art (e.g., discussed in more detailbelow). For example, the volume and type(s) of buffer employed duringthe use of the MCCS, MCCS1, and/or MCCS2 in any of the processesdescribed herein can be chosen in order to optimize one or more of thefollowing in the purified recombinant protein (e.g., recombinant proteindrug product): the overall yield of recombinant protein, the activity ofthe recombinant protein, the level of purity of the recombinant protein,and the removal of biological contaminants from a fluid (e.g., liquidculture medium) including the recombinant protein (e.g., absence ofactive viruses, mycobacteria, yeast, bacteria, or mammalian cells).

The MCCS, MCCS1, and/or MCCS2 can be a periodic counter currentchromatography system (PCCS). A PCCS can, e.g., include two or morechromatography columns (e.g., three columns or four columns) that areswitched in order to allow for the continuous elution of recombinantprotein from the two or more chromatography columns. A PCCS can includetwo or more chromatography columns, two or more chromatographicmembranes, or at least one chromatographic column and at least onechromatographic membrane. A column operation (cycle) generally consistsof the load, wash, eluate, and regeneration steps. In PCCSs, multiplecolumns are used to run the same steps discretely and continuously in acyclic fashion. Since the columns are operated in series, the flowthrough and wash from one column is captured by another column. Thisunique feature of PCCSs allows for loading of the resin close to itsstatic binding capacity instead of to the dynamic binding capacity, asis typical during batch mode chromatography. As a result of thecontinuous cycling and elution, fluid entering a PCCS is processedcontinuously, and the eluate including recombinant protein iscontinuously produced.

Column-switching strategy is employed to advance from one step toanother in a PCCS cycle. Examples of column switching that can be usedin a PCCS are described in U.S. Provisional Patent Application Ser. Nos.61/775,060 and 61/856,390. For example, a column switching method canemploy two automated switching operations per column: the first of whichis related to the initial product breakthrough, while the secondcoincides with column saturation. The determination of when the columnswitching operations should take place can be determined by monitoringthe recombinant protein concentration (e.g., monitoring performed by UVmonitoring) in the eluate from each chromatography column present in aPCCS. For example, column switching can be determined by any PAT toolcapable of in-line measurement of product concentration with feedbackcontrol. The PAT tool is capable of real-time in-line measurement ofproduct concentration with feedback control. As in known in the art,column switches can also be designed based on time or the amount offluid (e.g., buffer) passed through the one or more chromatographycolumn(s) and/or chromatographic membranes in the MCCS, MCCS1, and/orMCCS2.

In PCCSs, the residence time (RT) of the recombinant protein on the eachchromatography column and/or chromatographic membrane present in thePCCS can be decreased without increasing the column/membrane sizebecause the breakthrough from the first column/membrane can be capturedon another column/membrane in the PCCS. A continuous process system canbe designed to process liquid culture medium at any perfusion rate (D)by varying the column/membrane volume (V) and RT using the equation of:V=D*RT.

The one or more unit operations that can be performed by the MCCS or theMCC1 and/or MCCS2 used in the presently described processes include, forexample, capturing the recombinant protein, inactivating viruses presentin a fluid including the recombinant protein, purifying the recombinantprotein, polishing the recombinant protein, holding a fluid includingthe recombinant protein (e.g., using any of the exemplary break tank(s)described herein), filtering or removing particulate material and/orcells from a fluid including the recombinant protein, and adjusting theionic concentration and/or pH of a fluid including the recombinantprotein.

In some embodiments, the MCCS or the MCCS1 includes at least onechromatographic column and/or chromatographic membrane that performs theunit operation of capturing the recombinant protein. The unit operationof capturing can be performed using at least one chromatography columnand/or chromatography resin, e.g., that utilizes a capture mechanism.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Capturing can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,molecular sieve chromatography, or hydrophobic interactionchromatography. Non-limiting resins that can be used to capture arecombinant protein are described herein. Additional examples of resinsthat can be used to capture a recombinant protein are known in the art.

The unit operation of inactivating viruses present in a fluid includingthe recombinant protein can be performed using a MCCS, MCCS1, and/orMCCS2 (e.g., that include(s), e.g., a chromatography column, achromatography membrane, or a holding tank that is capable of incubatinga fluid including the recombinant protein at a pH of between about 3.0to 5.0 (e.g., between about 3.5 to about 4.5, between about 3.5 to about4.25, between about 3.5 to about 4.0, between about 3.5 to about 3.8, orabout 3.75) for a period of at least 30 minutes (e.g., a period ofbetween about 30 minutes to 1.5 hours, a period of between about 30minutes to 1.25 hours, a period of between about 0.75 hours to 1.25hours, or a period of about 1 hour).

The unit operation of purifying a recombinant protein can be performedusing one or more MCCSs (e.g., a MCCS, MCCS1, and/or MCCS2) thatinclude(s), e.g., a chromatography column or chromatographic membranethat includes a resin, e.g., that utilizes a capture system.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Purifying can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,molecular sieve chromatography, or hydrophobic interactionchromatography. Non-limiting resins that can be used to purify arecombinant protein are described herein. Additional examples of resinsthat can be used to purify a recombinant protein are known in the art.

The unit operation of polishing a recombinant protein can be performedusing one or more MCCSs (e.g., a MCCS, MCCS1, and/or MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat includes a resin, e.g., that can be used to perform cationexchange, anion exchange, molecular sieve chromatography, or hydrophobicinteraction chromatography. Non-limiting resins that can be used topolish a recombinant protein are described herein. Additional examplesof resins that can be used to polish a recombinant protein are known inthe art.

The unit operation of holding a fluid including the recombinant proteincan be performed using an MCCS (e.g., a MCCS, MCCS1, and/or MCCS2) thatincludes at least one reservoir (e.g., a break tank) or a maximum of 1,2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in the MCCS or theMCCS1 and MCCS2 combined. For example, the reservoir(s) (e.g., breaktank(s)) that can be used to achieve this unit operation can each have avolume of between about 1 mL to about 1 L (e.g., between about 1 mL toabout 800 mL, between about 1 mL to about 600 mL, between about 1 mL toabout 500 mL, between about 1 mL to about 400 mL, between about 1 mL toabout 350 mL, between about 1 mL to about 300 mL, between about 10 mLand about 250 mL, between about 10 mL and about 200 mL, between about 10mL and about 150 mL, or between about 10 mL to about 100 mL). Thereservoir(s) (e.g., break tank(s)) used in the processes describedherein can have a capacity that is, e.g., between 1 mL and about 300 mL,inclusive, e.g., between 1 mL and about 280 mL, about 260 mL, about 240mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, about 140mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL,about 20 mL, or about 10 mL, inclusive. Any of the reservoir(s) (e.g.,break tank(s)) used (in any of the processes described herein) to holdfluid before it enters into the MCCS or MCCS1 can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, between about 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the MCCS or MCCS1. Any of the reservoir(s) (e.g.,break tanks(s)) used to hold a eluate from MCCS1 (including therecombinant protein) before it enters the MCCS2 can have a capacity thatis, e.g., between 1 mL and about 100%, inclusive, e.g., between about 1mL and about 90%, about 80%, about 70%, about 60%, about 50%, about 40%,about 30%, about 20%, about 10%, or about 5%, inclusive, of the loadingvolume of the first column of the MCCS2.

The reservoir(s) (e.g., break tank(s)) can each hold the fluid includingthe recombinant protein for at least 10 minutes (e.g., at least 20minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 4 hours, or at least 6 hours). In other examples, the reservoir(s)(e.g., break tank(s)) only holds a recombinant protein for a total timeperiod of, e.g., between about 5 minutes and less than about 6 hours,inclusive, e.g., between about 5 minutes and about 5 hours, about 4hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes,inclusive. The reservoir(s) (e.g., break tank(s)) can be used to bothhold and refrigerate (e.g., at a temperature of less than 25° C., lessthan 15° C., or less than 10° C.) the fluid including the recombinantprotein. The reservoir can have any shape, including a circularcylinder, an oval cylinder, or an approximately rectangular sealed andnonpermeable bag.

The unit operations of filtering a fluid including the recombinantprotein can be performed using an MCCS (e.g., the MCCS, MCCS1, and/orMCCS2) that includes, e.g., a filter, or a chromatography column orchromatographic membrane that includes a molecular sieve resin. As isknown in the art, a wide variety of submicron filters (e.g., a filterwith a pore size of less than 1 μm, less than 0.5 μm, less than 0.3 μm,about 0.2 μm, less than 0.2 μm, less than 100 nm, less than 80 nm, lessthan 60 nm, less than 40 nm, less than 20 nm, or less than 10 nm) areavailable in the art that are capable of removing any precipitatedmaterial and/or cells (e.g., precipitated, unfolded protein;precipitated, unwanted host cell proteins; precipitated lipids;bacteria; yeast cells; fungal cells; mycobacteria; and/or mammaliancells). Filters having a pore size of about 0.2 μm or less than 0.2 μmare known to effectively remove bacteria from the fluid including therecombinant protein. As is known in the art, a chromatography column ora chromatographic membrane including a molecular sieve resin can also beused in an MCCS (e.g., the MCCS, MCCS1, and/or MCCS2) to perform theunit operation of filtering a fluid including a recombinant protein.

The unit operations of adjusting the ionic concentration and/or pH of afluid including the recombinant protein can be performed using a MCCS(e.g., a MCCS, a MCCS1, and/or a MCCS2) that includes and utilizes abuffer adjustment reservoir (e.g., an in-line buffer adjustmentreservoir) that adds a new buffer solution into a fluid that includesthe recombinant protein (e.g., between columns within the MCCS, MCCS1,and/or MCCS2, or after the last column in a penultimate MCCS (e.g., theMCCS1) and before the fluid including the recombinant protein is fedinto the first column of the next MCCS (e.g., the MCCS2)). As can beappreciated in the art, the in-line buffer adjustment reservoir can beany size (e.g., greater than 100 mL) and can include any bufferedsolution (e.g., a buffered solution that has one or more of: anincreased or decreased pH as compared to the fluid including therecombinant protein, an increased or decreased ionic (e.g., salt)concentration compared to the fluid including the recombinant protein,and/or an increased or decreased concentration of an agent that competeswith the recombinant protein for binding to resin present in at leastone chromatographic column or at least one chromatographic membrane inan MCCS (e.g., the MCCS, MCCS1, and/or MCCS2)).

The MCCS, MCCS1, and/or MCCS2 can perform two or more unit operations.For example, the MCCS, MCCS1, and/or MCCS2 can each perform at least thefollowing unit operations: capturing the recombinant protein andinactivating viruses present in the fluid including the recombinantprotein; capturing the recombinant protein, inactivating viruses presentin the fluid including the recombinant protein, and adjusting the ionicconcentration and/or pH of a liquid including the recombinant protein;purifying the recombinant protein and polishing the recombinant protein;purifying the recombinant protein, polishing the recombinant protein,and filtering a fluid including the recombinant protein or removingprecipitates and/or particular matter from a fluid including therecombinant protein; and purifying the recombinant protein, polishingthe recombinant protein, filtering a fluid including the recombinantprotein or removing precipitates and/or particulate matter from a fluidincluding the recombinant protein, and adjusting the ionic concentrationand/or pH of a liquid including the recombinant protein.

Capturing the Recombinant Protein

The present processes include a step of capturing the recombinantprotein using a MCCS or MCCS1. As can be appreciated in the art, theliquid culture medium including the recombinant protein can becontinuously fed onto the MCCS or MCCS1 using a variety of differentmeans. For example, the liquid culture medium can be actively pumpedinto the MCCS or MCCS1, or the liquid culture medium can be fed into theMCCS or MCCS1 using gravitational force. The liquid culture medium canbe stored in a reservoir (e.g., a holding tank) before it is fed intothe MCCS or MCCS1 or the liquid culture medium can be actively pumpedfrom a bioreactor including a culture of cells (e.g., mammalian cellsthat secrete the recombinant protein into the culture medium) into theMCCS or MCCS1.

The liquid culture medium can be fed (loaded) into the MCCS or MCCS1 ata flow rate of between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 1.0 mL/minute and about 15.0 mL/minute). Theliquid culture medium including the recombinant protein can be derivedfrom any of the exemplary sources described herein or known in the art.

Some examples further include the optional step of filtering the liquidculture medium before it is fed into the MCCS or MCCS1. Any of theexemplary means of filtering a liquid culture medium or a fluidincluding the recombinant protein described herein, or any filtrationmeans known in the art, can be used to filter the liquid culture mediumincluding the recombinant protein before it is fed into the MCCS orMCCS1.

In the processes described herein, the capturing of the recombinantprotein from the liquid culture medium is performed using the MCCS orMCCS1. As can be appreciated in the art, in order to achieve the captureof the recombinant protein, at least one chromatographic column or atleast one chromatographic membrane in the MCCS or MCCS1 must include aresin that utilizes a capturing mechanism (e.g., any of the exemplarycapturing mechanisms described herein), or includes a resin capable ofperforming cation exchange, anion exchange, molecular sieve, orhydrophobic interaction chromatography. For example, if the recombinantprotein is an antibody or an antibody fragment, the capturing system canbe a protein A-binding capturing mechanism or an antigen-bindingcapturing mechanism (where the capturing antigen is specificallyrecognized by the recombinant antibody or antibody fragment). If therecombinant protein is an enzyme, the capturing mechanism can use anantibody or antibody fragment that specifically binds to the enzyme tocapture the recombinant enzyme, a substrate of the enzyme to capture therecombinant enzyme, a cofactor of the enzyme to capture the recombinantenzyme, or, if the recombinant enzyme includes a tag, a protein, metalchelate, or antibody (or antibody fragment) that specifically binds tothe tag present in the recombinant enzyme. Non-limiting resins that canbe used to capture a recombinant protein are described herein andadditional resins that can be used to capture a recombinant protein areknown in the art. One non-limiting example of resin that utilizes aprotein A-binding capture mechanism is MabSelect SuRe resin (GEHealthcare, Piscataway, N.J.).

Exemplary non-limiting sizes and shapes of the chromatography column(s)or chromatographic membrane(s) present in the MCCS or MCCS1 that can beused to capture the recombinant protein are described herein. The liquidculture medium fed (loaded) into the MCCS or MCCS1 can include, e.g.,between about 0.05 mg/mL to about 100 mg/mL recombinant protein (e.g.,between about 0.1 mg/mL to about 90 mg/mL, between about 0.1 mg/mL toabout 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL, between about0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL to about 50 mg/mL,between about 0.1 mg/mL to about 40 mg/mL, between about 0.1 mg/mL toabout 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL,between about 0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL toabout 10 mg/mL, or between about 0.5 mg/mL to about 10 mg/mL recombinantprotein). The mean time required for the recombinant protein to bind tothe resin used to perform the unit operation of capturing can be, e.g.,between about 5 seconds to about 10 minutes (e.g., between about 10seconds to about 8 minutes, between about 10 seconds to about 7 minutes,between about 10 seconds to about 6 minutes, between about 10 seconds toabout 5 minutes, between about 30 seconds to about 5 minutes, betweenabout 1 minute to about 5 minutes, between about 10 seconds to about 4minutes, between about 30 seconds to about 4 minutes, or between about 1minute to about 4 minutes).

As can be appreciated in the art, in order to capture the recombinantprotein using the chromatography column(s) or chromatographicmembrane(s) present in the MCCS or MCCS1, one must perform thesequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the MCCS or MCCS1. Any of the exemplary flow rates, buffervolumes, and/or lengths of time allotted for each sequentialchromatographic step described herein can be used in the one or more ofthese different sequential chromatographic steps (e.g., one or more ofthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the MCCS or MCCS1 that are used for capturing the recombinantprotein). Non-limiting flow rates, buffer volumes, and/or lengths oftime allotted for each sequential chromatographic step that can be usedfor capturing chromatographic column(s) and/or chromatographicmembrane(s) in the MCCS or MCCS1 (e.g., a PCCS or PCCS1) are providedbelow. In addition, exemplary buffers that can be used in the MCCSand/or MCCS1 are described below.

The MCCS or MCCS1 including at least one chromatographic column and/orchromatographic membrane including a resin that can perform the unitoperation of capturing (e.g., any of exemplary resins that can be usedfor capturing described herein) can be loaded with the liquid culturemedium including a recombinant protein using any of loading flow rates(fed rates) described above. In some examples, a single chromatographiccolumn or single chromatographic membrane including a resin that iscapable of performing the unit operation of capturing is loaded in,e.g., between about 10 minutes to about 90 minutes (e.g., between about15 minutes and about 90 minutes, between about 20 minutes and 80minutes, between about 30 minutes and 80 minutes, between about 40minutes and about 80 minutes, between about 50 minutes and about 80minutes, and between about 60 minutes and 80 minutes). In some examples,wherein the MCCS or MCCS1 includes at least two chromatographic columnsthat include a resin that is capable of performing the unit operation ofcapturing in series, the time required to load two of thechromatographic columns in series is, e.g., between about 50 minutes toabout 180 minutes (e.g., between about 60 minutes and about 180 minutes,between about 70 minutes and about 180 minutes, between about 80 minutesand about 180 minutes, between about 90 minutes and about 180 minutes,between about 100 minutes and about 180 minutes, between about 110minutes and 150 minutes, and between about 125 minutes and about 145minutes).

Following the loading of the recombinant protein onto the at least onechromatographic column or chromatographic membrane in the MCCS or MCCS1that includes a resin that is capable of performing the unit operationof capturing, the at least one chromatographic column or chromatographicmembrane is washed with at least one washing buffer. As can beappreciated in the art, the at least one (e.g., two, three, or four)washing buffer is meant to elute all proteins that are not therecombinant protein from the at least one chromatography column orchromatographic membrane, while not disturbing the interaction of therecombinant protein with the resin.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 4×CV to about11×CV, about 5×CV to about 11×CV, or about 5×CV to about 10×CV). Thetotal time of the washing can be, e.g., between about 2 minutes to about3 hours (e.g., between about 2 minutes to about 2.5 hours, between about2 minutes to about 2.0 hours, between about 5 minutes to about 1.5hours, between about 10 minutes to about 1.5 hours, between about 10minutes to about 1.25 hours, between about 20 minutes to about 1.25hours, or between about 30 minutes to about 1 hour).

Following the washing of the at least one chromatographic column orchromatographic membrane in the MCCS or MCCS1 that includes a resin thatis capable of performing the unit operation of capturing, therecombinant protein is eluted from the at least one chromatographiccolumn or chromatographic membrane by passing an elution buffer throughthe at least one chromatographic column or chromatographic membrane inthe MCCS or MCCS1 that includes a resin that is capable of performingthe unit operation of capturing. The elution buffer can be passedthrough the at least one chromatography column or chromatographicmembrane that includes a resin that is capable of performing the unitoperation of capturing at a flow rate of between about 0.2 mL/minute toabout 25 mL/minute (e.g., between about 0.2 mL/minute to about 20mL/minute, between about 0.5 mL/minute to about 20 mL/minute, betweenabout 0.2 mL/minute to about 15 mL/minute, between about 0.5 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 1.0 mL/minute and about 15.0 mL/minute). Thevolume of elution buffer used to elute the recombinant protein from eachof the at least one chromatographic column or chromatographic membraneincluding a resin that is capable of performing the unit operation ofpurifying can be, e.g., between about 1× column volume (CV) to about15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV to about13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CVto about 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV,about 5×CV to about 11×CV, or about 5×CV to about 10×CV). The total timeof the eluting can be, e.g., between about 2 minutes to about 3 hours(e.g., between about 2 minutes to about 2.5 hours, between about 2minutes to about 2.0 hours, between about 2 minutes to about 1.5 hours,between about 2 minutes to about 1.5 hours, between about 2 minutes toabout 1.25 hours, between about 2 minutes to about 1.25 hours, betweenabout 2 minutes to about 1 hour, between about 2 minutes and about 40minutes, between about 10 minutes and about 40 minutes, between about 20minutes and about 40 minutes). Non-limiting examples of elution buffersthat can be used in these methods will depend on the capture mechanismand/or the recombinant protein. For example, an elution buffer caninclude a different concentration of salt (e.g., increased saltconcentration), a different pH (e.g., an increased or decreased saltconcentration), or a molecule that will compete with the recombinantprotein for binding to the resin that is capable of performing the unitoperation of capturing. Examples of such elution buffers for eachexemplary capture mechanism described herein are well known in the art.

Following the elution of the recombinant protein from the at least onechromatographic column or chromatographic membrane in the MCCS or theMCCS1 that includes a resin that is capable of performing the unitoperation of capturing, and before the next volume of liquid culturemedium can be loaded onto the at least one chromatographic column orchromatographic membrane, the at least one chromatography column orchromatographic membrane must be equilibrated using an regenerationbuffer. The regeneration buffer can be passed through the at least onechromatography column or chromatographic membrane that includes a resinthat is capable of performing the unit operation of capturing at a flowrate of, e.g., between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 5.0mL/minute to about 15.0 mL/minute, or between about 1.0 mL/minute andabout 15.0 mL/minute). In some examples, the regeneration buffer is adenaturing buffer (e.g., any of the denaturing buffers or combinationsof denaturing buffers described herein). The volume of regenerationbuffer used to equilibrate the at least one chromatography column orchromatographic membrane that includes a resin that is capable ofperforming the unit operation of capturing can be, e.g., between about1× column volume (CV) to about 15×CV (e.g., between about 1×CV to about14×CV, about 1×CV to about 13×CV, about 1×CV to about 12×CV, about 1×CVto about 11×CV, about 2×CV to about 11×CV, about 3×CV to about 11×CV,about 2×CV to about 5×CV, about 4×CV to about 11×CV, about 5×CV to about11×CV, or about 5×CV to about 10×CV).

In some of the processes described herein, the MCCS or MCCS1 includes areservoir that holds a fluid including the recombinant protein at low pH(e.g., a pH below 4.6, below 4.4, below 4.2, below 4.0, below 3.8, below3.6, below 3.4, below 3.2, or below 3.0) for, e.g., about 1 minute to1.5 hours (e.g., about 1 hour), and inactivates the viruses present in afluid including the recombinant protein. An example of a reservoir thatcan be used to perform the unit operation of inactivating viruses is astir flask (e.g., 500-mL stir flask, e.g., a 500-mL stir flask with aprogrammed stir plate) that is capable of holding a fluid including arecombinant protein for, e.g., about 1 minute to 1.5 hours, e.g., beforethe fluid including the recombinant protein is fed into the MCCS2. Thereservoir that is used to perform the unit operation of inactivation ofviruses can be a 500-mL stir flask with a programmed stir plate (e.g., astir plate programmed to mix (e.g., periodically mix) the fluid withinthe reservoir, e.g., every 4 hours). Another example of a reservoir thatcan be used to perform the unit operation of inactivation of viruses isa plastic bag (e.g., 500-mL plastic bag) that is capable of holding afluid including a recombinant protein for, e.g., about 1 minute to 1.5hours, e.g., before the fluid including the recombinant protein is fedinto the MCCS2. In some examples, the fluid including the recombinantprotein can already have a low pH (e.g., a pH below 4.6, below 4.4,below 4.2, below 4.0, below 3.8, below 3.6, below 3.4, below 3.2, orbelow 3.0) when it is fed into the reservoir that is used to perform theunit operation of viral inactivation. As can be appreciated by thoseskilled in the art, a variety of other means can be used to perform theunit operation of inactivating viruses. For example, UV irradiation of afluid including the recombinant protein can also be used to perform theunit operation of inactivating viruses. Non-limiting examples ofreservoirs that can be used to perform the unit operation ofinactivation of viruses present in a fluid including the recombinantprotein are described herein.

The MCCS or MCCS1 can include a PCCS including four chromatographycolumns, where at least three of the four chromatography columns performthe unit operation of capturing the recombinant protein from the liquidculture medium (e.g., using an MCCS that includes any of the at leastone chromatography columns that include a resin that is capable ofperforming the unit operation of capturing (e.g., any of those describedherein)). In these examples, the fourth-column of the PCC can performthe unit operation of inactivating viruses in a fluid that includes therecombinant protein (e.g., any of the exemplary columns described hereinthat can be used to achieve viral inactivation of a fluid including therecombinant protein).

In some examples, a fluid including the recombinant protein iscontinuously eluted from the MCCS1 (e.g., PCCS1), and is continuouslyfed into the MCCS2 (e.g., PCCS2). The percent of the recombinant proteinrecovered in the eluate of the MCCS or MCCS1 (e.g., PCCS or PCCS1) canbe, e.g., at least 70%, at least 72%, at least 74%, at least 76%, atleast 78%, at least 80%, at least 82%, at least 84%, at least 86%, atleast 88%, at least 90%, at least 92%, at least 94%, at least 96%, or atleast 98%). The eluate from the MCCS1 (e.g., PCCS1) can be fed into theMCCS2 (e.g., PCCS2) using a variety of means known in the art (e.g.,tubing). The eluate of the MCCS1 (e.g., PCCS1) can be fed into the MCCS2(e.g., PCCS2) at a flow rate of, e.g., between about 0.2 mL/minute toabout 25 mL/minute (e.g., between about 0.2 mL/minute to about 20mL/minute, between about 0.5 mL/minute to about 20 mL/minute, betweenabout 0.2 mL/minute to about 15 mL/minute, between about 0.5 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 5.0 mL/minute to about 15.0 mL/minute, betweenabout 15 mL/minute to about 25 mL/minute, or between about 1.0 mL/minuteand about 15.0 mL/minute).

Some processes described herein can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the MCCS1 (e.g.,PCCS1) before it is fed into the MCCS2 (e.g., PCCS2). As describedherein, the ionic concentration and/or pH of the eluate from the MCCS1(e.g., PCCS1) can be adjusted (before it is fed into the MCCS2) byadding a buffer to the eluate (e.g., through the use of an in-linebuffer adjustment reservoir). The buffer can be added to the eluate fromthe MCCS1 at a flow rate of, e.g., between about 0.1 mL/minute to about15 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, or between about0.5 mL/minute to about 5 mL/minute).

The processes described herein can further include a step of holding orstoring (and optionally also refrigerating) the eluate from the MCCS1prior to feeding the eluate from the MCCS1 into the MCCS2. As describedherein, this holding or storing step can be performed using any of thereservoirs (e.g., back-up tanks) described herein.

The processes described herein can also include a step of filtering theeluate from the MCCS1 before the eluate is fed into the MCCS2. Any ofthe exemplary filters or methods for filtration described herein can beused to filter the eluate from the MCCS1 before the eluate is fed intothe MCCS2.

Polishing and Purifying the Recombinant Protein

The MCCS, MCCS1, and/or MCCS2 can be used to perform the unit operationof purifying and polishing the recombinant protein. For example, theMCCS2 can be used to perform the operation of purifying and polishingthe recombinant protein and the eluate from the MCCS2 is a protein drugsubstance. The MCCS, MCCS1, and/or MCCS2 can include at least one (e.g.,two, three, or four) chromatography column or chromatographic membranethat can be used to perform the unit operation of purifying arecombinant protein, and at least one (e.g., two, three, or four)chromatography column or chromatographic membrane that can be used toperform the unit operation of polishing the recombinant protein.

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of purifying the recombinantprotein can include a resin that utilizes a capture mechanism (e.g., anyof the capture mechanisms described herein or known in the art), or aresin that can be used to perform anion exchange, cation exchange,molecular sieve, or hydrophobic interaction chromatography. The at leastone chromatography column or chromatographic membrane that can be usedto perform the unit of operation of polishing the recombinant proteincan include a resin that can be used to perform anion exchange, cationexchange, molecular sieve, or hydrophobic interaction chromatography(e.g., any of the exemplary resins for performing anion exchange, cationexchange, molecular sieve, or hydrophobic interaction chromatographydescribed herein or known in the art).

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit ofoperation of purifying the recombinant protein, and/or the size andshape of the at least one chromatographic membrane that can be used toperform the unit of operation of polishing the recombinant protein canany of combination of the exemplary sizes, shapes, and volumes ofchromatography columns or chromatographic membranes described herein. Ascan be appreciated by one skilled in the art, the step of purifying orpolishing a recombinant protein can, e.g., include the steps of loading,washing, eluting, and equilibrating the at least one chromatographycolumn or chromatographic membrane used to perform the unit of operationof purifying or polishing the recombinant protein. Typically, theelution buffer coming out of a chromatography column or chromatographicmembrane used to perform the unit operation of purifying includes therecombinant protein. Typically, the loading and/or wash buffer comingout of a chromatography column or chromatographic membrane used toperform the unit operation of polishing includes the recombinantprotein.

For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform unit operation ofpurifying the recombinant protein can have a volume of, e.g., betweenabout 2.0 mL to about 200 mL (e.g., between about 2.0 mL to about 180mL, between about 2.0 mL to about 160 mL, between about 2.0 mL to about140 mL, between about 2.0 mL to about 120 mL, between about 2.0 mL toabout 100 mL, between about 2.0 mL to about 80 mL, between about 2.0 mLto about 60 mL, between about 2.0 mL to about 40 mL, between about 5.0mL to about 40 mL, between about 2.0 mL to about 30 mL, between about5.0 mL to about 30 mL, or between about 2.0 mL to about 25 mL). The flowrate of the fluid including the recombinant protein as it is loaded ontothe at least one chromatography column or at least one chromatographicthat can be used to perform the unit operation of purifying therecombinant protein can be, e.g., between about 0.1 mL/minute to about25 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 0.1 mL/minute to about 2mL/minute, or about 0.2 mL/minute to about 4 mL/minute). Theconcentration of the recombinant protein in the fluid loaded onto the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinant proteincan be, e.g., between about 0.05 mg/mL to about 100 mg/mL recombinantprotein (e.g., between about 0.1 mg/mL to about 90 mg/mL, between about0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL,between about 0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL toabout 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL, between about0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL to about 20 mg/mL,between 0.5 mg/mL to about 20 mg/mL, between about 0.1 mg/mL to about 15mg/mL, between about 0.5 mg/mL to about 15 mg/mL, between about 0.1mg/mL to about 10 mg/mL, or between about 0.5 mg/mL to about 10 mg/mLrecombinant protein). The resin in the at least one chromatographycolumn or chromatographic membrane used to perform unit operation ofpurifying can be a resin that can be used to perform anion exchange orcation exchange chromatography. The resin in the at least onechromatography column or chromatographic membrane that is used toperform the unit operation of purifying can be a cationic exchange resin(e.g., Capto-S resin, GE Healthcare Life Sciences, Piscataway, N.J.).

Following the loading of the recombinant protein onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant protein, the atleast one chromatographic column or chromatographic membrane is washedwith at least one washing buffer. As can be appreciated in the art, theat least one (e.g., two, three, or four) washing buffer is meant toelute all proteins that are not the recombinant protein from the atleast one chromatography column or chromatographic membrane, while notdisturbing the interaction of the recombinant protein with the resin orotherwise eluting the recombinant protein.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 4×CV to about11×CV, about 2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about5×CV to about 10×CV). The total time of the washing can be, e.g.,between about 2 minutes to about 3 hours (e.g., between about 2 minutesto about 2.5 hours, between about 2 minutes to about 2.0 hours, betweenabout 5 minutes to about 1.5 hours, between about 10 minutes to about1.5 hours, between about 10 minutes to about 1.25 hours, between about20 minutes to about 1.25 hours, between about 30 minutes to about 1hour, between about 2 minutes and 10 minutes, between about 2 minutesand 15 minutes, or between about 2 minutes and 30 minutes).

Following the washing of the at least one chromatographic column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein, the recombinant protein is eluted from the atleast one chromatographic column or chromatographic membrane by passingan elution buffer through the at least one chromatographic column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein. The elution buffer can be passed through the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of purifying the recombinant proteinat a flow rate of between about 0.2 mL/minute to about 25 mL/minute(e.g., between about 0.2 mL/minute to about 20 mL/minute, between about0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minute toabout 15 mL/minute, between about 0.5 mL/minute to about 15 mL/minute,between about 0.5 mL/minute to about 10 mL/minute, between about 0.5mL/minute and about 6.0 mL/minute, between about 1.0 mL/minute and about5.0 mg/minute, between about 0.5 mL minute and about 14 mL/minute,between about 1.0 mL/minute and about 25.0 mL/minute, or between about1.0 mL/minute and about 15.0 mL/minute). The volume of elution bufferused to elute the recombinant protein from each the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant protein can be,e.g., between about 1× column volume (CV) to about 25×CV (e.g., betweenabout 1×CV to about 20×CV, between about 15×CV and about 25×CV, betweenabout 1×CV to about 14×CV, about 1×CV to about 13×CV, about 1×CV toabout 12×CV, about 1×CV to about 11×CV, about 2×CV to about 11×CV, about3×CV to about 11×CV, about 4×CV to about 11×CV, about 5×CV to about11×CV, or about 5×CV to about 10×CV). The total time of the eluting canbe, e.g., between about 2 minutes to about 3 hours (e.g., between about2 minutes to about 2.5 hours, between about 2 minutes to about 2.0hours, between about 2 minutes to about 1.5 hours, between about 2minutes to about 1.5 hours, between about 2 minutes to about 1.25 hours,between about 2 minutes to about 1.25 hours, between about 2 minutes toabout 1 hour, between about 2 minutes and about 40 minutes, betweenabout 10 minutes and about 40 minutes, between about 20 minutes andabout 40 minutes, or between about 30 minutes and 1.0 hour).Non-limiting examples of elution buffers that can be used in thesemethods will depend on the resin and/or the biophysical properties ofthe recombinant protein. For example, an elution buffer can include adifferent concentration of salt (e.g., increased salt concentration), adifferent pH (e.g., an increased or decreased salt concentration), or amolecule that will compete with the recombinant protein for binding tothe resin. Examples of such elution buffers for each of the exemplarycapture mechanisms described herein are well known in the art.

Following the elution of the recombinant protein from the at least onechromatographic column or chromatographic membrane used to perform theunit operation of purifying the recombinant protein, and before the nextvolume of fluid including a recombinant protein can be loaded onto theat least one chromatographic column or chromatographic membrane, the atleast one chromatography column or chromatographic membrane must beequilibrated using an regeneration buffer. The regeneration buffer canbe passed through the at least one chromatography column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein at a flow rate of, e.g., between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL/minute and about 6.0 mL/minute, betweenabout 1.0 mL/minute and about 5.0 mg/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 5.0 mL/minute to about 15.0 mL/minute, orbetween about 1.0 mL/minute and about 15.0 mL/minute). The volume ofregeneration buffer used to equilibrate the at least one chromatographycolumn or chromatographic membrane that includes a resin that can beused to perform the unit operation of purifying the recombinant proteincan be, e.g., between about 1× column volume (CV) to about 15×CV (e.g.,between about 1×CV to about 14×CV, between about 1×CV to about 13×CV,between about 1×CV to about 12×CV, between about 1×CV to about 11×CV,between about 2×CV to about 11×CV, between about 3×CV to about 11×CV,between about 2×CV to about 5×CV, between about 2.5×CV to about 7.5×CV,between about 4×CV to about 11×CV, between about 5×CV to about 11×CV, orbetween about 5×CV to about 10×CV). The concentration of recombinantprotein in the eluate of the at least one chromatography column orchromatographic membrane used to perform the unit operation of purifyingthe recombinant protein can be, e.g., between about 0.05 mg/mL to about100 mg/mL recombinant protein (e.g., between about 0.1 mg/mL to about 90mg/mL, between about 0.1 mg/mL to about 80 mg/mL, between about 0.1mg/mL to about 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL,between about 0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL toabout 40 mg/mL, between about 2.5 mg/mL and about 7.5 mg/mL, betweenabout 0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL to about 20mg/mL, between 0.5 mg/mL to about 20 mg/mL, between about 0.1 mg/mL toabout 15 mg/mL, between about 0.5 mg/mL to about 15 mg/mL, between about0.1 mg/mL to about 10 mg/mL, or between about 0.5 mg/mL to about 10mg/mL recombinant protein).

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of polishing the recombinantprotein can include a resin that can be used to perform cation exchange,anion exchange, or molecular sieve chromatography. As can be appreciatedin the art, polishing a recombinant protein using the at least onechromatography column or chromatography membrane that can be used toperform the unit operation of polishing the recombinant protein caninclude, e.g., the steps of loading, chasing, and regenerating the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing the recombinant protein.For example, when the steps of loading, chasing, and regenerating areused to perform the polishing, the recombinant protein does not bind theresin in the at least one chromatography column or chromatographymembrane that is used to perform the unit operation of polishing therecombinant protein, and the recombinant protein is eluted from the atleast one chromatography column or chromatographic membrane in theloading and chasing steps, and the regenerating step is used to removeany impurities from the at least one chromatography column orchromatographic membrane before additional fluid including therecombinant protein can be loaded onto the at least one chromatographycolumn or chromatographic membrane. Exemplary flow rates and buffervolumes to be used in each of the loading, chasing, and regeneratingsteps are described below.

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit operationof polishing the recombinant protein, and/or the size and shape of theat least one chromatographic membrane that can be used to perform theunit operation of polishing the recombinant protein can any ofcombination of the exemplary sizes, shapes, and volumes ofchromatography columns or chromatographic membranes described herein.For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein can have a volume of, e.g., betweenabout 0.5 mL to about 200 mL (e.g., between about 0.5 mL to about 180mL, between about 0.5 mL to about 160 mL, between about 0.5 mL to about140 mL, between about 0.5 mL to about 120 mL, between about 0.5 mL toabout 100 mL, between about 0.5 mL to about 80 mL, between about 0.5 mLto about 60 mL, between about 0.5 mL to about 40 mL, between about 5.0mL to about 40 mL, between about 0.5 mL to about 30 mL, between about5.0 mL to about 30 mL, between about 0.5 mL to about 25 mL, betweenabout 0.2 mL to about 10 mL, or between about 0.2 mL to about 5 mL). Theflow rate of the fluid including the recombinant protein as it is loadedonto the at least one chromatography column or chromatographic membranethat can be used to perform the unit operation of polishing therecombinant protein can be, e.g., between about 0.1 mL/minute to about25 mL/minute (e.g., between about 0.1 mL/minute to about 12.5 mL/minute,between about 0.1 mL/minute to about 10.0 mL/minute, between about 0.1mL/minute to about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 2 mL/minute and about 6mL/minute, between about 0.1 mL/minute to about 2 mL/minute, or about0.2 mL/minute to about 4 mL/minute). The total volume of fluid includinga recombinant protein loaded onto the at least one chromatography columnor chromatographic membrane that can be used to perform the unitoperation of polishing the recombinant protein can be, e.g., betweenabout 1.0 mL to about 250 mL (e.g., between about 1.0 mL to about 225mL, between about 1.0 mL to about 200 mL, between about 1.0 mL to about175 mL, between about 1.0 mL to about 150 mL, between about 100 mL toabout 125 mL, between about 100 mL to about 150 mL, between about 1.0 mLto about 150 mL, between about 1.0 mL to about 125 mL, between about 1.0mL to about 100 mL, between about 1.0 mL to about 75 mL, between about1.0 mL to about 50 mL, or between about 1.0 mL to about 25 mL). Theresin in the at least one chromatography column or chromatographicmembrane used to perform the polishing can be an anion exchange orcation exchange resin. The resin in the at least one chromatographycolumn or chromatographic membrane that is used to perform the unitoperation of polishing can be a cationic exchange resin (e.g.,Sartobind® Q resin, Sartorius, Goettingen, Germany).

Following the loading step, a chasing step is performed (e.g., a chasebuffer is passed through the at least one chromatography membrane orchromatographic membrane to collect the recombinant protein which doesnot substantially bind to the at least one chromatography column orchromatographic membrane). In these examples, the chase buffer can bepassed through the at least one chromatography column or chromatographicmembrane at a flow rate of between about 0.2 mL/minute to about 50mL/minute (e.g., between about 1 mL/minute to about 40 mL/minute,between about 1 mL/minute to about 30 mL/minute, between about 5mL/minute to about 45 mL/minute, between about 10 mL/minute to about 40mL/minute, between about 0.2 mL/minute to about 20 mL/minute, betweenabout 0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute). The volume of chase buffer used can be, e.g., between about1× column volume (CV) to about 100×CV (e.g., between about 1×CV to about90×CV, between about 1×CV to about 80×CV, between about 1×CV to about70×CV, between about 1×CV to about 60×CV, between about 1× to about50×CV, between about 1×CV to about 40×CV, between about 1×CV to about30×CV, between about 1×CV to about 20×CV, between about 1×CV to about15×CV, between about 5×CV to about 20×CV, between about 5×CV to about30×CV, between about 1×CV to about 14×CV, about 1×CV to about 13×CV,about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CV toabout 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV, about2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about 5×CV toabout 10×CV). The total time of the chasing can be, e.g., between about1 minute to about 3 hours (e.g., between about 1 minute to about 2.5hours, between about 1 minute to about 2.0 hours, between about 1minutes to about 1.5 hours, between about 2 minutes to about 1.5 hours,between about 1 minutes to about 1.25 hours, between about 2 minutes toabout 1.25 hours, between about 1 minute to about 5 minutes, betweenabout 1 minute to about 10 minutes, between about 2 minutes to about 4minutes, between about 30 minutes to about 1 hour, between about 2minutes and 10 minutes, between about 2 minutes and 15 minutes, orbetween about 2 minutes and 30 minutes). The combined concentration ofrecombinant protein present in the eluate coming through the column inthe loading step and the chasing step can be, e.g., between about 0.1mg/mL to about 100 mg/mL recombinant protein (e.g., between about 0.1mg/mL to about 90 mg/mL, between about 0.1 mg/mL to about 80 mg/mL,between about 0.1 mg/mL to about 70 mg/mL, between about 0.1 mg/mL toabout 60 mg/mL, between about 0.1 mg/mL to about 50 mg/mL, between about0.1 mg/mL to about 40 mg/mL, between about 2.5 mg/mL and about 7.5mg/mL, between about 0.1 mg/mL to about 30 mg/mL, between about 0.1mg/mL to about 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL, betweenabout 0.1 mg/mL to about 15 mg/mL, between about 0.5 mg/mL to about 15mg/mL, between about 0.1 mg/mL to about 10 mg/mL, between about 0.5mg/mL to about 10 mg/mL, or between about 1 mg/mL and about 5 mg/mLrecombinant protein).

Following the chasing step and before the next volume of fluid includingthe recombinant protein can be loaded onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of polishing, the at least one chromatographycolumn or chromatographic membrane must be regenerated using aregeneration buffer. The regeneration buffer can be passed through theat least one chromatography column or chromatographic membrane that canbe used to perform the unit operation of polishing the recombinantprotein at a flow rate of, e.g., between about 0.2 mL/minute to about 50mL/minute (e.g., between about 1 mL/minute to about 40 mL/minute,between about 1 mL/minute to about 30 mL/minute, between about 5mL/minute to about 45 mL/minute, between about 10 mL/minute to about 40mL/minute, between about 0.2 mL/minute to about 20 mL/minute, betweenabout 0.5 mL/minute to about 20 mL/minute, between about 0.2 mL/minuteto about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, or between about 1.0 mL/minute and about 15.0mL/minute). The volume of regeneration buffer used to regenerate the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing can be, e.g., betweenabout 1× column volume (CV) to about 500×CV (e.g., between about 1×CV toabout 450×CV, between about 1×CV to about 400×CV, between about 1×CV toabout 350×CV, between about 1×CV to about 300×CV, between about 1×CV toabout 250×CV, between about 1×CV to about 200×CV, between about 1×CV toabout 150×CV, between about 1×CV to about 100×CV, between about 1×CV toabout 90×CV, between about 1×CV to about 80×CV, between about 1×CV toabout 70×CV, between about 1×CV to about 60×CV, between about 1× toabout 50×CV, between about 1×CV to about 40×CV, between about 1×CV toabout 30×CV, between about 1×CV to about 20×CV, between about 1×CV toabout 15×CV, between about 5×CV to about 20×CV, between about 5×CV toabout 30×CV, between about 1×CV to about 14×CV, about 1×CV to about13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CVto about 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV,about 2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about 5×CVto about 10×CV).

In other examples, the one or more chromatography column(s) and/orchromatographic membranes used to perform the unit operation ofpolishing include a resin that selectively binds or retains theimpurities present in a fluid including the recombinant protein, andinstead of regenerating the one or more column(s) and/or membrane(s),the one or more column(s) and/or membrane(s) are replaced (e.g.,replaced with a substantially similar column(s) and/or membrane(s)) oncethe binding capacity of the resin in the one or more column(s) and/ormembrane(s) has been reached or is substantially close to being reached.

In some examples of these processes described herein, the MCCS2 includesa PCCS including three chromatography columns and one chromatographicmembrane, e.g., where the three chromatography columns in the PCCSperform the unit operation of purifying the recombinant protein (e.g.,using at least one chromatography column(s) that can be used to performthe unit of operation of purifying the protein) and the chromatographicmembrane in the PCCS performs the unit operation of polishing therecombinant protein. In these examples, the chromatographic membrane inthe PCCS that can be used to perform the unit operation of polishing thetherapeutic protein can be any of the exemplary chromatographicmembranes described herein that can be used to perform the unitoperation of polishing the recombinant protein. Any of the columnswitching methods described herein can be used to determine when thefirst three chromatography columns and the chromatographic membrane inthe PCCS in this example can be switched.

Some embodiments of this example can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the threechromatographic columns in the PCCS before the eluate is fed into thechromatographic membrane in the PCCS. As described herein, the ionicconcentration and/or pH of the eluate from the three chromatographycolumns in PCCS can be adjusted (before it is fed into thechromatographic membrane in the PCCS in this example)) by adding abuffer to the eluate of the three chromatography columns in the PCCS(e.g., through the use of an in-line buffer adjustment reservoir). Thebuffer can be added to the eluate at a flow rate of, e.g., between about0.1 mL/minute to about 15 mL/minute (e.g., between about 0.1 mL/minuteto about 12.5 mL/minute, between about 0.1 mL/minute to about 10.0mL/minute, between about 0.1 mL/minute to about 8.0 mL/minute, betweenabout 0.1 mL/minute to about 6 mL/minute, between about 0.1 mL/minute to4 mL/minute, or between about 0.5 mL/minute to about 5 mL/minute).

These examples can further include a step of holding or storing theeluate from the three chromatography columns in the PCCS in this exampleprior to feeding the eluate into the chromatographic membrane(chromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein). As described herein, this holdingor storing step can be performed using any of the reservoirs (e.g.,back-up tanks) described herein.

These examples can also include a step of filtering the eluate from thechromatographic membrane in the exemplary PCCS system (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein). Any of the exemplary filters ormethods for filtration described herein can be used to filter the eluatefrom the chromatographic membrane in this exemplary PCCS (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant protein).

As can be appreciated by those in the art, the purified recombinantprotein can be periodically eluted from the MCCS or MCCS2 using any ofthe processes described herein. For example, any of the processesdescribed herein can elute the purified recombinant protein for aduration of, e.g., between about 30 seconds and about 5 hours (e.g.,between about 1 minute and about 4 hours, between about 1 minute andabout 3 hours, between about 1 minute and about 2 hours, between about 1minute or about 1.5 hours, between about 1 minute and about 1 hour, orbetween about 1 minute and about 30 minutes) at a frequency of, e.g.,between about 1 minute and about 6 hours (e.g., between about 1 minuteand about 5 hours, between about 1 minute and about 4 hours, betweenabout 1 minute and about 3 hours, between about 1 minute and 2 hours,between about 1 minute and 1 hour, or between about 1 minute and 30minutes), depending on, e.g., the chromatography column(s) and/orchromatographic membrane(s) used in the MCCS or the MCCS1 and MCCS2.

Culturing Methods

Some of the processes described herein further include a step ofculturing cells (e.g., recombinant mammalian cells) that secrete arecombinant protein in a bioreactor (e.g., a perfusion or fed-batchbioreactor) that includes a liquid culture medium, wherein a volume ofthe liquid culture medium that is substantially free of cells (e.g.,mammalian cells) is continuously or periodically removed from thebioreactor (e.g., perfusion bioreactor) and fed into the MCCS or MCCS1.The bioreactor can have a volume of, e.g., between about 1 L to about10,000 L (e.g., between about 1 L to about 50 L, between about 50 L toabout 500 L, between about 500 L to about 1000 L, between 500 L to about5000 L, between about 500 L to about 10,000 L, between about 5000 L toabout 10,000 L, between about 1 L and about 10,000 L, between about 1 Land about 8,000 L, between about 1 L and about 6,000 L, between about 1L and about 5,000 L, between about 100 L and about 5,000 L, betweenabout 10 L and about 100 L, between about 10 L and about 4,000 L,between about 10 L and about 3,000 L, between about 10 L and about 2,000L, or between about 10 L and about 1,000 L). The amount of liquidculture medium present in a bioreactor can be, e.g., between aboutbetween about 0.5 L to about 5,000 L (e.g., between about 0.5 L to about25 L, between about 25 L to about 250 L, between about 250 L to about500 L, between 250 L to about 2500 L, between about 250 L to about 5,000L, between about 2500 L to about 5,000 L, between about 0.5 L and about5,000 L, between about 0.5 L and about 4,000 L, between about 0.5 L andabout 3,000 L, between about 0.5 L and about 2,500 L, between about 50 Land about 2,500 L, between about 5 L and about 50 L, between about 5 Land about 2,000 L, between about 5 L and about 1,500 L, between about 5L and about 1,000 L, or between about 5 L and about 500 L). Culturingcells can be performed, e.g., using a fed-batch bioreactor or aperfusion bioreactor. Non-limiting examples and different aspects ofculturing cells (e.g., culturing mammalian cells) are described belowand can be used in any combination.

Cells

The cells that are cultured in some of the processes described hereincan be bacteria (e.g., gram negative bacteria), yeast (e.g.,Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, orArxula adeninivorans), or mammalian cells. The mammalian cell can be acell that grows in suspension or an adherent cell. Non-limiting examplesof mammalian cells that can be cultured in any of the processesdescribed herein include: Chinese hamster ovary (CHO) cells (e.g., CHODG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (e.g., NS/0),B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells(e.g., HEK 293E and HEK 293F), African green monkey kidney epithelialcells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidneyepithelial cells (MDCK) cells. In some examples where an adherent cellis cultured, the culture can also include a plurality of microcarriers(e.g., microcarriers that include one or more pores). Additionalmammalian cells that can be cultured in any of the processes describedherein are known in the art.

The mammalian cell can include a recombinant nucleic acid (e.g., anucleic acid stably integrated in the mammalian cell's genome) thatencodes a recombinant protein (e.g., a recombinant protein).Non-limiting examples of recombinant nucleic acids that encode exemplaryrecombinant proteins are described below, as are recombinant proteinsthat can be produced using the methods described herein. In someinstances, the mammalian cell that is cultured in a bioreactor (e.g.,any of the bioreactors described herein) was derived from a largerculture.

A nucleic acid encoding a recombinant protein can be introduced into amammalian cell using a wide variety of methods known in molecularbiology and molecular genetics. Non-limiting examples includetransfection (e.g., lipofection), transduction (e.g., lentivirus,adenovirus, or retrovirus infection), and electroporation. In someinstances, the nucleic acid that encodes a recombinant protein is notstably integrated into a chromosome of the mammalian cell (transienttransfection), while in others the nucleic acid is integrated.Alternatively or in addition, the nucleic acid encoding a recombinantprotein can be present in a plasmid and/or in a mammalian artificialchromosome (e.g., a human artificial chromosome). Alternatively or inaddition, the nucleic acid can be introduced into the cell using a viralvector (e.g., a lentivirus, retrovirus, or adenovirus vector). Thenucleic acid can be operably linked to a promoter sequence (e.g., astrong promoter, such as a β-actin promoter and CMV promoter, or aninducible promoter). A vector including the nucleic acid can, ifdesired, also include a selectable marker (e.g., a gene that confershygromycin, puromycin, or neomycin resistance to the mammalian cell).

In some instances, the recombinant protein is a secreted protein and isreleased by the mammalian cell into the extracellular medium (e.g., thefirst and/or second liquid culture medium). For example, a nucleic acidsequence encoding a soluble recombinant protein can include a sequencethat encodes a secretion signal peptide at the N- or C-terminus of therecombinant protein, which is cleaved by an enzyme present in themammalian cell, and subsequently released into the extracellular medium(e.g., the first and/or second liquid culture medium).

Culture Media

Liquid culture media are known in the art. The liquid culture media(e.g., a first and/or second tissue culture medium) can be supplementedwith a mammalian serum (e.g., fetal calf serum and bovine serum), and/ora growth hormone or growth factor (e.g., insulin, transferrin, andepidermal growth factor). Alternatively or in addition, the liquidculture media (e.g., a first and/or second liquid culture medium) can bea chemically-defined liquid culture medium, an animal-derived componentfree liquid culture medium, a serum-free liquid culture medium, or aserum-containing liquid culture medium. Non-limiting examples ofchemically-defined liquid culture media, animal-derived component freeliquid culture media, serum-free liquid culture media, andserum-containing liquid culture media are commercially available.

A liquid culture medium typically includes an energy source (e.g., acarbohydrate, such as glucose), essential amino acids (e.g., the basicset of twenty amino acids plus cysteine), vitamins and/or other organiccompounds required at low concentrations, free fatty acids, and/or traceelements. The liquid culture media (e.g., a first and/or second liquidculture medium) can, if desired, be supplemented with, e.g., a mammalianhormone or growth factor (e.g., insulin, transferrin, or epidermalgrowth factor), salts and buffers (e.g., calcium, magnesium, andphosphate salts), nucleosides and bases (e.g., adenosine, thymidine, andhypoxanthine), protein and tissue hydrolysates, and/or any combinationof these additives.

A wide variety of different liquid culture media that can be used toculture cells (e.g., mammalian cells) in any of the methods describedherein are known in the art. Medium components that also may be usefulin the present processes include, but are not limited to,chemically-defined (CD) hydrolysates, e.g., CD peptone, CD polypeptides(two or more amino acids), and CD growth factors. Additional examples ofliquid tissue culture medium and medium components are known in the art.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium described herein can be thesame type of media or different media.

Additional Features of Exemplary Bioreactors

The interior surface of any of the bioreactors described herein may haveat least one coating (e.g., at least one coating of gelatin, collagen,poly-L-ornithine, polystyrene, and laminin), and as is known in the art,one or more ports for the sparging of O₂, CO₂, and N₂ into the liquidculture medium, and a stir mechanism for agitating the liquid culturemedium. The bioreactor can incubate the cell culture in a controlledhumidified atmosphere (e.g., at a humidity of greater than 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or a humidity of 100%).The bioreactor can also be equipped with a mechanical device that iscapable of removing a volume of liquid culture medium from thebioreactor and optionally, a filter within the mechanical device thatremoves the cells from the liquid culture medium during the process oftransfer of the liquid culture medium out of the bioreactor (e.g., anATF system or the cell filtering system described in U.S. ProvisionalPatent Application Ser. No. 61/878,502).

Temperature

The step of culturing of mammalian cells can be performed at atemperature of about 31° C. to about 40° C. Skilled practitioners willappreciate that the temperature can be changed at specific time point(s)in during the culturing step, e.g., on an hourly or daily basis. Forexample, the temperature can be changed or shifted (e.g., increased ordecreased) at about one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, eleven days,twelve days, fourteen days, fifteen days, sixteen days, seventeen days,eighteen days, nineteen days, or about twenty days or more after theinitial seeding of the bioreactor with the cell (e.g., mammalian cell).For example, the temperature can be shifted upwards (e.g., a change ofup to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20°C.). For example, the temperature can be shifted downwards (e.g., achange of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to orabout 20° C.).

CO₂

The culturing step described herein can further include exposing theliquid culture medium in the bioreactor to an atmosphere including atmost or about 15% CO₂ (e.g., at most or about 14% CO₂, 12% CO₂, 10% CO₂,8% CO₂, 6% CO₂, 5% CO₂, 4% CO₂, 3% CO₂, 2% CO₂, or at most or about 1%CO₂).

Perfusion Bioreactor

The culturing step described herein can be performed using a perfusionbioreactor. Culturing a cell (e.g., a mammalian cell) in a perfusionbioreactor includes the removal from the bioreactor of a first volume ofa first liquid culture medium (e.g., including any concentration ofmammalian cells, e.g., a first volume of a first liquid culture mediumthat is substantially free of cells), and adding to the first liquidculture medium a second volume of a second liquid culture medium.Removal and adding can be performed simultaneously or sequentially, or acombination of the two. Further, removal and adding can be performedcontinuously (e.g., at a rate that removes and replaces a volume ofbetween 0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%,between 1% and 500%, between 1% and 400%, between 1% and 350%, between1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and200%, between 5% and 150%, between 10% and 50%, between 15% and 40%,between 8% and 80%, or between 4% and 30%) of the volume of thebioreactor or the first liquid culture medium volume over any given timeperiod (e.g., over a 24-hour period, over an incremental time period ofabout 1 hour to about 24 hours, or over an incremental time period ofgreater than 24 hours)) or periodically (e.g., once every third day,once every other day, once a day, twice a day, three times a day, fourtimes a day, or five times a day), or any combination thereof. Whereperformed periodically, the volume that is removed or replaced (e.g.,within about a 24-hour period, within an incremental time period ofabout 1 hour to about 24 hours, or within an incremental time period ofgreater than 24 hours) can be, e.g., between 0.1% to 800% (e.g., between1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and400%, between 1% and 300%, between 1% and 200%, between 1% and 100%,between 100% and 200%, between 5% and 150%, between 10% and 50%, between15% and 40%, between 8% and 80%, or between 4% and 30%) of the volume ofthe bioreactor or the first liquid culture medium volume. The firstvolume of the first liquid culture medium removed and the second volumeof the second liquid culture medium added can in some instances be heldapproximately the same over each 24-hour period (or, alternatively, anincremental time period of about 1 hour to about 24 hours or anincremental time period of greater than 24 hours) over the entire orpart of the culturing period. As is known in the art, the rate at whichthe first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can bevaried. The rate at which the first volume of the first liquid culturemedium is removed (volume/unit of time) and the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be about the same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 1 hour and about 24 hours or an incremental timeperiod of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the bioreactor volume or the firstliquid culture medium volume to about 25% to about 150% of thebioreactor volume or the first liquid culture medium volume.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different.

The first volume of the first liquid culture medium can be removed,e.g., by a mechanical system that can remove the first volume of thefirst liquid culture medium from the bioreactor (e.g., the first volumeof the first liquid culture medium that is substantially free of cellsfrom the bioreactor). Alternatively or in addition, the first volume ofthe first liquid culture medium can be removed by seeping or gravityflow of the first volume of the first liquid culture medium through asterile membrane with a molecular weight cut-off that excludes the cell(e.g., mammalian cell).

The second volume of the second liquid culture medium can be added tothe first liquid culture medium in an automated fashion, e.g., byperfusion pump.

In some instances, removing the first volume of the first liquid culturemedium (e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells) and adding to the first liquidculture medium a second volume of the second liquid culture medium doesnot occur within at least 1 hour (e.g., within 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours) of the seeding of the bioreactor with a mammalian cell.

Fed-Batch Bioreactor

The culturing step described herein can be performed using a fed-batchbioreactor. Culturing a cell in a fed-batch bioreactor includes, overthe majority of the culturing period, the addition (e.g., periodic orcontinuous addition) to the first liquid culture medium of a secondvolume of a second liquid culture medium. The adding of the secondliquid culture medium can be performed continuously (e.g., at a ratethat adds a volume of between 0.1% to 300% (e.g., between 1% and 250%,between 1% and 100%, between 100% and 200%, between 5% and 150%, between10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and30%) of the volume of the bioreactor or the first liquid culture mediumvolume over any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isadded (e.g., within about a 24-hour period, within an incremental timeperiod of about 1 hour to about 24 hours, or within an incremental timeperiod of greater than 24 hours) can be, e.g., between 0.1% to 300%(e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%,between 5% and 150%, between 10% and 50%, between 15% and 40%, between8% and 80%, or between 4% and 30%) of the volume of the bioreactor orthe first liquid culture medium volume. The second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be varied over the entire or part of the culturing period. Forexample, the volume of the second liquid culture medium added can change(e.g., gradually increase) over each 24-hour period (or alternatively,an incremental time period of between 1 hour and about 24 hours or anincremental time period of greater than 24 hours) during the culturingperiod. For example the volume of the second liquid culture medium addedwithin each 24-hour period (or alternatively, an incremental time periodof between about 1 hour and above 24 hours or an incremental time periodof greater than 24 hours) over the culturing period can be increased(e.g., gradually or through staggered increments) over the culturingperiod from a volume that is between 0.5% to about 20% of the bioreactorvolume or the first liquid culture medium volume to about 25% to about150% of the bioreactor volume or the first liquid culture medium volume.The rate at which the second volume of the second liquid culture mediumis added (volume/unit of time) can be about the same over the entire orpart of the culturing period.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different. The volume of the secondliquid culture medium can be added to the first liquid culture medium inan automated fashion, e.g., by perfusion pump.

In some instances, adding to the first liquid culture medium a secondvolume of the second liquid culture medium does not occur within atleast 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours,within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9hours, within 10 hours, within 12 hours, within 14 hours, within 16hours, within 18 hours, within 24 hours, within 36 hours, within 48hours, within 72 hours, within 96 hours, or after 96 hours) of theseeding of the bioreactor with a mammalian cell. The cell culture mediumin fed-batch cultures is typically harvested at the end of cultureperiod and used in any of the processes described herein, however, thecell culture medium in fed-batch cultures can also be harvested at oneor more time points during the culturing period and used in any of theprocesses described herein.

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., containers, volumes, rates or frequencies of replacingculture volumes, agitation frequencies, temperatures, media, and CO₂concentrations) can be used in any combination in to perform thesemethods. Further, any of the mammalian cells described herein or knownin the art can be used to produce a recombinant protein.

Exemplary Biological Manufacturing Systems

Examples of biological manufacturing systems useful for performing theprocesses described herein and that include a MCCS or a MCCS1 and MCCS2are described in U.S. Provisional Patent Application Ser. Nos.61/775,060 and 61/856,390 (incorporated by reference). In theseexemplary systems, at least one (e.g., at least two, three, four, five,or six) chromatography column including a gamma-irradiatedchromatography resin is present in the MCCS or in the MCCS1 and/orMCCS2. For example, the entire system can include a total of two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twentychromatography columns including a gamma-irradiated chromatographyresin. For example, the MCCS, MCCS1, and/or MCCS2 can include (or caneach include) one, two, three, four, five, six, seven, eight, nine, orten chromatography columns including a gamma-irradiated chromatographyresin.

For example, useful systems can include a MCCS1 that includes an inletand a MCCS2 that includes an outlet, or an MCCS that includes an inletand an outlet. In some embodiments, the MCCS1 and MCCS2 are in fluidcommunication with each other. These systems can also be configured suchthat fluid can be passed into the inlet, through the MCCS1 and MCCS2,and exit the manufacturing system through the outlet. These systemsprovide for the continuous and time-efficient production of atherapeutic drug substance from a liquid culture medium. For example,the elapsed time between feeding a fluid (e.g., a liquid culture medium)including a therapeutic protein into the MCCS1 and eluting purifiedrecombinant protein (e.g., therapeutic protein drug substance) from theoutlet of the MCCS2 can be, e.g., between about 4 hours and about 48hours, inclusive.

Some exemplary systems do not include a break tank. In others, thesystem can include a maximum of 1, 2, 3, 4, or 5 break tank(s) in theentire system (e.g., where each break tank only holds a therapeuticprotein for a total time period of, e.g., between about 5 minutes andabout 6 hours, inclusive). The break tank(s) can have a capacity that isbetween 1 mL and about 300 mL, inclusive. Any break tank(s) disposed inthe system such that fluid enters the break tank(s) prior to enteringMCCS1 or MCCS can have a capacity that is between 1 mL and about 100%,inclusive, of the loading volume of the first column of the MCCS1 orMCCS, respectively. Any break tanks(s) disposed in the system such thatfluid enters the break tank(s) prior to entering the MCCS2 (and afterexiting the MCCS1) can have a capacity that is, e.g., between 1 mL andabout 100%, inclusive, of the loading volume of the first column of theMCCS2.

Additional Exemplary System Structures and Features

The MCCS or MCCS1 can include an inlet through which fluid (e.g., aliquid culture medium that is substantially free of cells) can be passedinto the MCCS or MCCS1, respectively. The inlet can be any structureknown in the art for such purposes. It can include, e.g., a threading,ribbing, or a seal that allows for a fluid conduit to be inserted, suchthat after insertion of the fluid conduit into the inlet, fluid willenter the MCCS or MCCS1 through the inlet without significant seepage offluid out of the inlet. Non-limiting inlets that can be used in thepresent systems are known and would be understood by those in the art.

The MCCS or MCCS1 can include at least two chromatography columns, atleast two chromatographic membranes, or at least one chromatographycolumn and at least one chromatographic membrane, and an inlet. The MCCSor MCCS1 can be any of the exemplary MCCSs described herein, or have oneor more of any of the exemplary features of an MCCS (in any combination)described herein. The chromatography column(s) and/or thechromatographic membrane(s) present in the MCCS or MCCS1 can have one ormore of any of the exemplary shapes, sizes, volumes (bed volumes),and/or unit operation(s) described herein.

The chromatography column(s) and/or the chromatographic membrane(s)present in the MCCS or MCCS1 can include one or more of any of theexemplary resins described herein or known in the art. For example, theresin included in one or more of the chromatography column(s) and/orchromatographic membrane(s) present in the MCCS or MCCS1 can be a resinthat utilizes a capture mechanism (e.g., protein A-binding capturemechanism, protein G-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, substrate-binding capture mechanism,cofactor-binding capture mechanism, an aptamer-binding capturemechanism, and/or a tag-binding capture mechanism). The resin includedin one or more of the chromatography column(s) and/or chromatographicmembrane(s) of the MCCS or MCCS1 can be a cation exchange resin, ananion exchange resin, a molecular sieve resin, or a hydrophobicinteraction resin, or any combination thereof. Additional examples ofresins that can be used to purify a recombinant protein are known in theart, and can be included in one or more of the chromatography column(s)and/or chromatographic membrane(s) present in the MCCS or MCCS1. Thechromatography column(s) and/or chromatography membranes present in theMCCS or MCCS1 can include the same and/or different resins (e.g., any ofthe resins described herein or known in the art for use in recombinantprotein purification).

The two or more chromatography column(s) and/or chromatographic resin(s)present in the MCCS or MCCS1 can perform one or more unit operations(e.g., capturing a recombinant protein, purifying a recombinant protein,polishing a recombinant protein, inactivating viruses, adjusting theionic concentration and/or pH of a fluid including the recombinantprotein, or filtering a fluid including a recombinant protein). Innon-limiting examples, the MCCS or MCCS1 can perform the unit operationsof capturing a recombinant protein from a fluid (e.g., a liquid culturemedium) and inactivating viruses present in the fluid including therecombinant protein. The MCCS or MCCS1 can perform any combinations oftwo of more unit operations described herein or known in the art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS or MCCS1 can be connected or moved with respect to eachother by a switching mechanism (e.g., a column-switching mechanism). TheMCCS or MCCS1 can also include one or more (e.g., two, three, four, orfive) pumps (e.g., automated, e.g., automated peristaltic pumps). Thecolumn-switching events can be triggered by the detection of a level ofrecombinant protein detected by UV absorbance corresponding to a certainlevel of recombinant protein in the fluid passing through the MCCS orMCCS1 (e.g., the input into and/or eluate from one or more of thechromatography column(s) and/or chromatographic membranes in the MCCS orMCCS1), a specific volume of liquid (e.g., buffer), or specific timeelapsed. Column switching generally means a mechanism by which at leasttwo different chromatography columns and/or chromatographic membranes inan MCCS or MCCS1 (e.g., two or more different chromatography columnsand/or chromatographic membranes present in the MCCS1 or MCCS2) areallowed to pass through a different step (e.g., equilibration, loading,eluting, or washing) at substantially the same time during at least partof the process.

The MCCS or MCCS1 can be a Periodic Counter-Current Chromatographysystem (PCCS). For example, the PCCS that is the MCCS or MCCS1 (i.e.,PCCS or PCCS1, respectively) can include four chromatography columns,where the first three columns perform the unit operation of capturing arecombinant protein from a fluid (e.g., a liquid culture medium), andthe fourth column of the PCCS performs the unit operation ofinactivating viruses in the fluid including the recombinant protein. APCCS that is the MCCS or MCCS1 can utilize a column-switching mechanism.The PCC system can utilize a modified ÄKTA system (GE Healthcare,Piscataway, N.J.) capable of running up to, e.g., four, five, six,seven, or eight columns, or more.

The MCCS or MCCS1 can be equipped with: one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV monitors, one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) valves,one or more (e.g., two, three, four, five, six, seven, eight, nine, orten) pH meters, and/or one or more (e.g., two, three, four, five, six,seven, eight, nine, or ten) conductivity meters. The MCCS or MCCS1 canalso be equipped with an operating system that utilizes software (e.g.,Unicorn-based software, GE Healthcare, Piscataway, N.J.) for sensingwhen a column-switching should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting (triggering) thecolumn-switching events.

The MCCS or MCCS1 can further include one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty,twenty-one, twenty-two, twenty-three, or twenty-four) in-line bufferadjustment reservoir(s) and/or a buffer reservoir(s). In other examples,the MCCS or MCCS1 can include one or more (e.g., two, three, four, five,or six) break tanks that can hold fluid that cannot readily pass intoone or more of the chromatography columns and/or chromatographicmembranes in the MCCS or MCCS1. The systems described herein can includeone or more break tanks (e.g., a break tank described herein) in theMCCS, MCCS1, and/or MCCS2. Other examples of the systems describedherein do not include a break tank in the MCCS, MCCS1, or MCCS2, or donot include a break tank in the entire system. Other examples of thesystems described herein include a maximum of one, two, three, four, orfive break tank(s) (e.g., any break tank(s) described herein) in theentire system.

Second MCCS

The second MCCS (MCCS2) in the exemplary systems includes at least twochromatography columns, at least two chromatographic membranes, or atleast one chromatography column(s) and at least one chromatographicmembrane(s), and an outlet. The MCCS2 can any of the exemplary MCCSsdescribed herein, or can have one or more of any of the exemplaryfeatures of an MCCS (in any combination) described herein. Thechromatography column(s) and/or the chromatographic membrane(s) presentin the MCCS2 can have one or more of: any of the shapes, sizes, volumes(bed volumes), and/or unit operations described herein. Thechromatography column(s) and/or the chromatographic membrane(s) caninclude any of the exemplary resins described herein or known in theart. For example, the resin included in one or more of thechromatography column(s) and/or chromatographic membrane(s) present inthe MCCS2 can be a resin that utilizes a capture mechanism (e.g.,protein A-binding capture mechanism, protein G-binding capturemechanism, antibody- or antibody fragment-binding capture mechanism,substrate-binding capture mechanism, cofactor-binding capture mechanism,tag-binding capture mechanism, and/or aptamer-binding capturemechanism). Useful resins include, e.g., a cation exchange resin, ananion exchange resin, a molecular sieve resin, and a hydrophobicinteraction resin. Additional examples of resins are known in the art.The chromatography column(s) and/or chromatography membranes present inthe MCCS2 can include the same and/or different resins (e.g., any of theresins described herein or known in the art for use in recombinantprotein purification).

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS2 can perform one or more unit operations (e.g., any of theunit operations described herein or any combination of the unitoperations described herein). In non-limiting examples, the MCCS2 canperform the unit operations of purifying a recombinant protein from afluid and polishing the recombinant protein present in the fluidincluding the recombinant protein. In other non-limiting examples, theMCCS2 can perform the unit operations of purifying a recombinant proteinpresent in a fluid, polishing a recombinant protein present in a fluid,and filtering a fluid including a recombinant protein. In anotherexample, the MCCS2 can perform the unit operations of purifying arecombinant protein present in a fluid, polishing a recombinant proteinpresent in a fluid, filtering a fluid including a recombinant protein,and adjusting the ionic concentration and/or pH of a fluid including arecombinant protein. The MCCS2 can perform any combination of two ofmore unit operations described herein or known in the art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the MCCS2 can be connected or moved with respect to each other by aswitching mechanism (e.g., a column-switching mechanism). The MCCS2 canalso include one or more (e.g., two, three, four, or five) pumps (e.g.,automated, e.g., automated peristaltic pumps). The column-switchingevents can be triggered by the detection of a level of recombinantprotein detected by UV absorbance corresponding to a certain level ofrecombinant protein in the fluid passing through the MCCS2 (e.g., theinput into and/or eluate from one or more of the chromatographycolumn(s) and/or chromatographic membranes in the MCCS2), a specificvolume of liquid (e.g., buffer), or specific time elapsed.

The MCCS2 be a Periodic Counter-Current Chromatography system (i.e.,PCCS2). For example, the PCCS2 can include three columns that performthe unit operation of purifying a recombinant protein from a fluid, anda chromatographic membrane that performs the unit operation of polishinga recombinant protein present in a fluid. For example, the three columnsthat perform the unit operation of purifying a recombinant protein froma fluid can include, e.g., a cationic exchange resin, and thechromatographic membrane that performs the unit operation of polishingcan include a cationic exchange resin. A PCCS2 can utilize acolumn-switching mechanism. The PCCS2 can utilize a modified ÄKTA system(GE Healthcare, Piscataway, N.J.) capable of running up to, e.g., four,five, six, seven, or eight columns, or more.

The MCCS2 can be equipped with: one or more (e.g., two, three, four,five, six, seven, eight, nine, or ten) UV monitors, one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) valves, one ormore (e.g., two, three, four, five, six, seven, eight, nine, or ten) pHmeters, and/or one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) conductivity meters. The MCCS2 can also be equippedwith an operating system that utilizes software (e.g., Unicorn-basedsoftware, GE Healthcare, Piscataway, N.J.) for sensing when acolumn-switching event should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting the column-switchingevents.

The MCCS2 can further include one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one,twenty-two, twenty-three, or twenty-four) in-line buffer adjustmentreservoir(s) and/or a buffer reservoir(s). In other examples, the MCCS2can include one or more (e.g., two, three, four, five, or six) breaktanks (e.g., any of the break tanks described herein) that can holdfluid that cannot readily pass into one or more of the chromatographycolumns and/or chromatographic membranes in the MCCS2.

The MCCS2 includes an outlet through which the therapeutic protein drugsubstance can exit the system. The outlet can include, e.g., athreading, ribbing, or a seal that allows for a fluid conduit to beinserted or a vial designed to hold or store the purified recombinantprotein (e.g., therapeutic protein drug substance). An outlet caninclude a surface that can be used to seal a reduced bioburden vial orother such storage container onto the outlet in order to allow thepurified recombinant protein (e.g., therapeutic protein drug substance)to flow directly into the reduced bioburden vial or storage container.Non-limiting outlets that can be used in the present systems are knownand would be understood by those in the art.

The systems described herein can also include a fluid conduit that isdisposed between the MCCS1 and the MCCS2. Any of the fluid conduitsdescribed herein can be, e.g., a tube that is made of, e.g.,polyethylene, polycarbonate, or plastic. The fluid conduit disposedbetween the MCCS1 and the MCCS2 can further include one of more of thefollowing in any combination: one or more in-line buffer adjustmentreservoirs that are in fluid communication with the fluid conduit andare positioned such that the buffer stored within the in-line bufferadjustment reservoir(s) is added to the fluid present in the fluidconduit; a break tank (e.g., any of the break tank(s) described herein)that is in fluid communication with the fluid conduit and is positionedsuch that it can hold any excess fluid present in the fluid conduit thatis unable to readily feed into the MCCS2; and one or more filters thatare disposed in the fluid conduit such that they are capable offiltering (e.g., removing bacteria) the fluid present in the fluidconduit. Any of the in-line buffer adjustment reservoirs can include,e.g., a volume of between about 0.5 L to 50 L of buffer (e.g., at atemperature at or below 25° C., 15° C., or 10° C.).

The systems described herein can optionally include a fluid conduitdisposed between the final chromatography column or chromatographicmembrane in the MCCS2 and the outlet. The systems described herein canfurther include one or more filters in fluid connection with the fluidconduit disposed between the final chromatography column orchromatographic membrane in the MCCS2 and the outlet, such that thefilter can remove, e.g., precipitated material, particulate matter, orbacteria from the fluid present in the fluid conduit disposed betweenthe final chromatography column or chromatographic membrane in the MCCS2and the outlet.

Some examples of the systems provided herein also include a bioreactorthat is in fluid connectivity with the inlet of the MCCS or MCCS1. Anyof the exemplary bioreactors described herein or known in the art can beused in the present systems.

Some examples of the systems provided herein also include a pump system.A pump system can include one or more the following: one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) pumps (e.g.,any of the pumps described herein or known in the art), one or more(e.g., two, three, four, or five) filters (e.g., any of the filtersdescribed herein or known in the art), one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV detectors, and one ormore (e.g., two, three, four, or five) break tanks (e.g., any of thebreak tanks described herein). Some examples of the systems providedherein further include a fluid conduit disposed between the pump and theinlet of the MCCS or MCCS1 (e.g., any of the exemplary fluid conduitsdescribed herein or known in the art). In some examples, this particularfluid conduit can include one or more (e.g., two, three, or four) pumps(e.g., any of the pumps described herein or known in the art) and/or oneor more (e.g., two, three, or four) break tanks (e.g., any of theexemplary break tanks described herein), where these pump(s) and/orbreak tank(s) are in fluid connection with the fluid present in thefluid conduit.

Some examples of the systems described herein further include a furtherfluid conduit connected to the fluid conduit between the pump and theinlet, where one end of the further fluid conduit is fluidly connectedto a bioreactor and the other end is fluidly connected to the fluidconduit between the pump and the inlet. This further fluid conduit caninclude a filter that is capable of removing cells from the liquidculture medium removed from the bioreactor (e.g., ATF cell retentionsystem).

The systems provided herein allow for the continuous production of apurified recombinant protein (e.g., therapeutic protein drug substance).As is known in the art, the systems can provide for the periodic elutionof a purified recombinant protein (e.g., therapeutic protein drugsubstance). The systems described herein can also result in a net yieldof purified recombinant protein (e.g., therapeutic protein drugsubstance) of at least about 5 g/day, at least about 10 g/day, at leastabout 15 g/day, at least about 20 g/day, at least about 30 g/day, or atleast about 40 g/day over a continuous period of at least about 5 days,at least about 10 days, at least about 15 days, at least about 20 days,at least about 25 days, at least about 30 days, at least about 40 days,at least about 50 days, at least about 60 days, at least about 70 days,at least about 80 days, at least about 90 days, or least about 100 days.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Examples Example 1 Effect of Gamma-Irradiation on Chromatography ResinBinding Capacity at T0

A set of experiments was performed to study the effect ofgamma-irradiation on the binding capacity of a bimodal chromatographyresin having both anionic exchange and hydrophobic properties (AEresin). The AE resin used in these experiments was Capto Adhere (GEHealthcare Life Sciences), which has a ligand of N-benzyl-N-methylethanolamine, an average particle size of 75 μm, and an ionic capacityof 0.09-0.12 mmol Cl⁻¹/mL medium. The AE resin was either left untreated(virgin) or treated to reduce the resin's bioburden (exposed to 25 kGygamma-irradiation). Irradiation was performed using 0.2 mL of resinslurried in 50 mM sodium phosphate, pH 7.0.

The amount of protein (Fabrazyme®) bound to untreated AE resin (inliquid culture medium) and 25 kGy gamma-irradiated AE resin at t0 (withno chromatography runs) was determined at different target protein(Fabrazyme®) concentrations. FIG. 1 shows the binding isotherms at t0for the untreated and 25 kGy gamma-irradiated AE resin. These data showthat the binding of target protein does not change aftergamma-irradiation of resin at to.

Example 2 Effect of Gamma-Irradiation on Chromatography Resin BindingCapacity Over Multiple Cycles and Use of Denaturing Buffers to Mitigatethe Loss in Binding Capacity

Experiments were performed to test the effect of gamma-irradiation onthe binding capacity of chromatography resin over multiple cycles. Inmulti-column chromatography, each of the columns used is loaded withprotein (Fabrazyme® in liquid culture medium) to its binding capacity.Column equilibration and washing was performed with 20 mM MES(2-(N-morpholino)ethanesulfonic acid), pH 7.0. Elution buffer, 200 mMarginine, 270 mM MES, 20% ethylene glycol pH 7.5 was used to elute thebound protein from the columns. In order to test the performance ofgamma-irradiated chromatography resin over its entire lifecycle in thisExample, columns containing untreated AE or 25 kGy gamma-irradiatedchromatography resin (prepared as described in Example 1) were cycledusing a multi-column chromatography system (MCCS) or single columns wereused to perform repeated cycles of chromatography under conditionsmimicking MCCS conditions (each column was loaded to its static bindingcapacity, before washing and elution were performed). Each of the washand eluting steps in the experiments described in this Example wereperformed in a step-wise fashion.

Multiple cycles of column chromatography were performed using a singlechromatography column including the AE resin (virgin, 15 kGygamma-irradiated, or 25 kGy gamma-irradiated resin) and a wash buffer of700 mM arginine, 100 mM acetate (pH 3.0), followed with a solution of 1N NaOH (after elution of the recombinant protein) in each cycle. Thepercentage of normalized binding capacity (as compared to the untreated(virgin) resin at t0) over multiple cycles was determined. The data fromthese experiments show that while the binding capacity for both theuntreated and gamma-irradiated resin are similar at t0, the bindingcapacity of the untreated and gamma-irradiated resin show a differentrate of decrease in binding capacity over multiple cycles ofchromatography (FIG. 2). While the virgin resin shows a decrease inbinding capacity over multiple cycles of chromatography, thegamma-irradiated resin shows a more dramatic decrease in bindingcapacity over multiple cycles of chromatography (FIG. 2). The calculatedrate of drop in binding capacity for each resin is this set ofexperiments is also shown in FIG. 3. These data demonstrate that the useof gamma-irradiation to reduce the bioburden of chromatography resultsin a decrease in the binding capacity of the resin that becomesprogressively worse over multiple cycles of chromatography.

Sets of experiments were performed to determine whether a variety ofdenaturing buffers could be used to recover the lost binding capacity ofgamma-irradiated chromatography resin. In these experiments, cycles ofchromatography performed using the virgin (untreated) AE resin wereperformed using a wash buffer of 700 mM arginine, 100 mM acetate (pH3.0), followed with a solution of 1 N NaOH (low pH arginine). Cycles ofchromatography were also performed using the 25 kGy gamma-irradiatedresin using one of the following combinations of buffers for washing theresin after elution of the recombinant protein in each cycle: 8 M urea,1 M NaCl, 0.1 M citric acid, pH 2.5, followed by a solution of 1 N NaOH(low pH urea); 6 M guanidine HCl (pH 2.5), followed by a solution of 1 NNaOH (or 1 M NaOH+1 M NaCl) (low pH guanidine); 0.5% Triton-X 100 in 0.1M acetic acid (pH 2.5), followed by a solution of 0.7 M acetic acid, 20%ethanol, 50% ethylene glycol (pH 2.5), followed by a solution of 1 NNaOH (low pH Triton); 0.7 M acetic acid, 20% ethanol, 50% ethyleneglycol (pH 2.5), followed by a solution of 1 N NaOH (low pH organic); or700 mM arginine, 100 mM acetate (pH 3.0), followed with a solution of 1N NaOH (low pH arginine). The normalized percentage binding capacity(normalized to the binding capacity of virgin (untreated) resin at t=0),the percentage recovery of recombinant protein (relative to the amountof recombinant protein present in the fluid loaded onto the resin) percycle, and the levels of host cell protein (ng/mg) present in the eluateper cycle were determined. The data in FIG. 4 show that all of testeddenaturing buffers were able to mitigate the loss in binding capacity ofthe gamma-irradiated chromatography resin, except for 0.7 M acetic acid,20% ethanol, 50% ethylene glycol (pH 2.5) and 700 mM arginine, 100 mMacetate (pH 3.0). The data in FIG. 4 show that all of the denaturingbuffers, except for 0.7 M acetic acid, 20% ethanol, 50% ethylene glycol(pH 2.5) and 700 mM arginine, 100 mM acetate (pH 3.0), are able toefficiently clean gamma-irradiated chromatography resin and maintain thebinding capacity of gamma-irradiated chromatography resin over multiplecycles of chromatography.

The data in FIG. 6 show that all of the tested denaturing buffers resultin a steady percentage recovery of the recombinant protein bound togamma-irradiated chromatography resin per cycle over multiple cycles.The data in FIG. 6 show that each of the tested denaturing buffers wasable to recover whatever amount of protein was bound to thegamma-irradiated chromatography resin per cycle to a similar extent. Thedata in FIG. 7 show that the use of the various tested denaturingbuffers result in acceptable levels of host cell protein in therecombinant protein eluate in each cycle.

The denaturing buffer used in each cycle was thought to recover thebinding capacity of the gamma-irradiated chromatography resin byreleasing tightly bound proteins from the resin. A representativechromatographs of multiple cycles of chromatography performed usingthree chromatography columns including 25 kGy gamma-irradiated AE resin,washed with 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5, and asolution of 1 N NaOH after elution of the recombinant protein (aftereach cycle) were recorded (FIG. 5). The chromatographs show that thetreatment of the gamma-irradiated resin with the denaturing bufferresulted in the release of a substantially the same amount of proteinfrom the gamma-irradiated resin in each of the three chromatographycolumns over multiple cycles (see arrows in FIG. 5).

In sum, these data show that different denaturing buffers can be used torecover the binding capacity of gamma-irradiated chromatography resin toachieve expected performance.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of performing chromatography withgamma-irradiated chromatography resin, comprising: (a) providing achromatography column comprising a gamma-irradiated chromatographyresin; (b) performing a first cycle of chromatography through thecolumn, wherein the cycle includes exposing the chromatography resin toa denaturing buffer; and (c) performing at least one additional cycle ofchromatography through the column.
 2. The method of claim 1, whereinperforming the cycles in (b) and/or (c) comprises the steps of: (a)capturing a recombinant protein by exposing the chromatography resinwith a liquid comprising a recombinant protein; (b) washing thechromatography resin by exposing the chromatography resin with a washbuffer; (c) eluting the recombinant protein by exposing thechromatography resin with an elution buffer; and (d) regenerating thechromatography resin by exposing the chromatography resin to thedenaturing buffer.
 3. The method of claim 1, wherein the cycles in steps(b) and (c) are performed using a closed and integrated system.
 4. Themethod of claim 3, wherein the denaturing buffer is a reduced bioburdenbuffer.
 5. The method of claim 1, wherein the denaturing buffercomprises one or more of the group consisting of urea, guanidinehydrochloride, and Triton™ X-100.
 6. The method of claim 1, wherein thecycle of step (b) further includes exposing the chromatography resin towash buffer comprising about 0.5 M to about 1.5 M sodium hydroxidefollowing exposure to the denaturing buffer.
 7. The method of claim 1,wherein the column is part of a multi-column chromatography system(MCCS).
 8. The method of claim 7, wherein the MCCS is a periodic countercurrent chromatography system (PCCS).
 9. The method of claim 1, whereinthe chromatography resin is an anion exchange chromatography resin, acation exchange chromatography resin, a size exclusion chromatographyresin, a hydrophobic interaction chromatography resin, an affinitychromatography resin, or any combination thereof.
 10. The method ofclaim 9, wherein the chromatography resin is an anion exchangechromatography resin.
 11. The method of claim 1, wherein thechromatography resin has been treated with a dose of gamma-irradiationbetween about 10 kGy to about 40 kGy.
 12. The method of claim 1, whereinfour or more additional cycles of chromatography are performed.
 13. Themethod of claim 1, wherein step (c) is performed continuously over aperiod of at least 4 days.
 14. The method of claim 2, wherein therecombinant protein is a therapeutic recombinant protein.
 15. Anintegrated, closed, and continuous process for manufacturing of apurified recombinant protein comprising: (a) providing a liquid culturemedium comprising a recombinant protein that is substantially free ofcells; and (b) continuously feeding the liquid culture medium into amulti-column chromatography system (MCCS) comprising at least onechromatography column comprising gamma-irradiated chromatography resin,wherein the chromatography resin is exposed during each cycle todenaturing buffer; wherein the process utilizes reduced bioburdenbuffer, is integrated, and runs continuously from the liquid culturemedium to an eluate from the MCCS that is the purified recombinantprotein.
 16. The process of claim 15, wherein the MCCS includes aplurality of columns for affinity chromatography, cation exchangechromatography, anion exchange chromatography, size exclusionchromatography, or hydrophobic interaction chromatography, or anycombination thereof.
 17. The process of claim 15, wherein therecombinant protein is a therapeutic recombinant protein.
 18. Theprocess of claim 15, wherein the denaturing buffer comprises one or moreof the group consisting of urea, guanidine hydrochloride, and Triton™X-100.
 19. The method of claim 15, wherein the chromatography resin isexposed to wash buffer comprising about 0.5 M to about 1.5 M sodiumhydroxide following exposure to denaturing buffer in each cycle.
 20. Anintegrated, closed, and continuous process for manufacturing of arecombinant protein comprising: (a) providing a liquid culture mediumcomprising a recombinant protein that is substantially free of cells;(b) continuously feeding the liquid culture medium into a firstmulti-column chromatography system (MCCS1); (c) capturing therecombinant protein from the liquid culture medium using the MCCS1; (d)producing an eluate from the MCCS1 that comprises the recombinantprotein and continuously feeding the eluate into a second multi-columnchromatography system (MCCS2); and (e) continuously feeding therecombinant protein from the eluate into the MCCS2 and subsequentlyeluting the recombinant protein to thereby produce the purifiedrecombinant protein, wherein: the process utilizes reduced bioburdenbuffer, is integrated, and runs continuously from the liquid culturemedium to the purified recombinant protein, and at least one column inthe MCCS1 and/or MCCS2 is a chromatography column comprisinggamma-irradiated chromatography resin and the chromatography resin isexposed to denaturing buffer during each cycle in the process.
 21. Theprocess of claim 20, wherein the MCCS1 further performs the unitoperations of capturing the recombinant protein and inactivating virusesand the MCCS2 performs the unit operations of purifying and polishingthe recombinant protein.
 22. The process of claim 20, wherein all of thechromatography column(s) in MCCS1 and MCCS2 are chromatography columnscomprising gamma-irradiated chromatography resin.
 23. The process ofclaim 20, wherein the MCCS1 is a first periodic counter currentchromatography system (PCCS 1) and the MCCS2 is a second periodiccounter current chromatography system.
 24. The process of claim 20,wherein the capturing is performed using affinity chromatography, cationexchange chromatography, anion exchange chromatography, or sizeexclusion chromatography, hydrophobic interaction chromatography, or anycombination thereof.
 25. The process of claim 20, wherein therecombinant protein is a therapeutic recombinant protein.
 26. Theprocess of claim 20, wherein the process is performed continuously for aperiod of at least 4 days.
 27. The process of claim 20, wherein thechromatography resin is an ion exchange chromatography resin, a cationexchange chromatography resin, a size exclusion chromatography resin, ahydrophobic interaction chromatography resin, an affinity chromatographyresin, or any combination thereof.
 28. The process of claim 20, whereinthe chromatography resin has been treated with a dose ofgamma-irradiation between about 10 kGy to about 40 kGy.
 29. The processof claim 20, wherein the denaturing buffer comprises one or more ofurea, guanidine hydrochloride, and Triton™ X-100.
 30. The method ofclaim 20, wherein the chromatography resin is exposed to a wash buffercomprising about 0.5 M to about 1.5 M sodium hydroxide followingexposure to the denaturing buffer.